Antibody neutralizers of human granulocyte macrophage colony stimulating factor

ABSTRACT

The present invention relates to a human monoclonal antibody or fragment thereof which specifically binds to and neutralizes primate GM-CSF.

CROSS REFERENCES TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/198,558, filed Aug. 4, 2011 (now U.S. Pat. No. 9,067,993, issued Jun.30, 2015), which is a continuation of U.S. patent application Ser. No.11/918,368, filed Oct. 12, 2007, which is the U.S. National Stage ofInternational Application No. PCT/EP2006/003528, filed Apr. 18, 2006,which claims benefit of European Patent Application No. 05008410.2,filed Apr. 18, 2005, each of which is hereby incorporated by reference.

The present invention relates to antibodies and fragments thereof whichneutralize the activity of human granulocyte macrophage colonystimulating factor (GM-CSF). The invention further related topharmaceutical compositions comprising such antibodies and fragmentsthereof as well as to uses of such antibodies and fragments thereof forthe preparation of medicaments for the treatment of various conditions.

Originally described as a potent stimulus of the growth anddifferentiations of granulocyte and macrophage precursor cells in vitro,granulocyte-macrophage colony-stimulating factor (GM-CSF) is anapproximately 23 kDa glycoprotein with a four alpha helical bundlestructure that binds to a heterodimeric receptor composed of subunitsbelonging to the type 1 cytokine receptor family. It stimulates thematuration of i.a. macrophages, neutrophils, granulocytes, eosinophilsand antigen-presenting dendritic cells, to increase their functionalcapacity in combating infections. Genetic ablation experiments i.e.experiments silencing or knocking out the gene of interest—hereGM-CSF—in mice indicating that GM-CSF is essential for maintaining thefunctional activity of some macrophage populations such as thoseinvolved in clearing surfactant in the lung and in responding to certainkinds of infection or immune responses.

While GM-CSF has potent stimulatory activity in vitro on progenitorcells for neutrophils, eosinophils, macrophages, and to a lesser extenterythroid and megakaryocytic cells, results obtained in vivo with geneknockout mice suggest that the major physiological role of GM-CSF is tomaining or stimulate the functional activity of mature macrophages andgranulocytes and to stimulate antigen presentation to the immune system.It does the latter by its director effects on dendritic cell andmacrophage production, but also by increasing expression of the class IImajor histocompatibility complex and Fc receptor on macrophages anddendritic cells.

GM-CSF stimulates the functional activities of neutrophils, eosinophils,and monocyte-macrophages. These include enhancement of chemotacticactivity, increased expression of cellular adhesion molecules andincreased adhesion to surfaces, and increased phagocytic activity aswell as inhibition and delay of apoptosis of these cells. Neutrophilsrepresent the first line of defence against aggressions. The programmeddeath of neutrophils is delayed by pro-inflammatory stimuli includingGM-CSF to ensure a proper resolution of the inflammation in time andplace. GM-CSF also stimulates the capacity of these cells to mediateantibody-dependent cell cytotoxicity and to kill microorganismsintracellularly and has a ‘priming’ effect on these cells to enhancetheir response to subsequent stimuli for the oxidative burst (superoxideanion production), degranulation and release of antimicrobial agents,and chemotaxis. Further, GM-CSF stimulates the release of secondarycytokines and mediators from these cells including IL-1, G-CSF, M-CSF,and leukotrienes from neutrophils, as well as IL-1, TNF, IL-6, G-CSF,M-CSF, and prostaglandins from macrophages.

It is clear from the above that GM-CSF plays a key role in activatingand maintaining the cell populations necessary to ward off infection.However, in some instances activation of these cell populations may beundesirable. For example, activation of the above cell lineages when nopathogen is present leads in many instances to acute and/or chronicinflammatory conditions which, in extreme cases, may belife-threatening. Similarly, over-expression of GM-CSF may lead toexcess immune activation, resulting in inflammation. In such instances,it may be desirable to neutralize the activity of GM-CSF such that thesymptoms of these inflammatory conditions are eliminated or at leastmitigated.

Examples of such neutralizing activity exist in the prior art. Forexample, it was found that a neutralizing anti-GM-CSF antibodycontributed to an increase in eosinophil apoptosis rate in peripheralblood samples (Kankaanranta et al. (2000) Journal of Allergy andClinical Immunology 106, 77-83). As enhanced eosinophil survival iscorrelated with asthma, an increase in eosinophil apoptosis would beexpected to mitigate asthmatic symptoms.

In chronic inflammatory diseases such as asthma, rheumatoid arthritis,and multiple sclerosis levels of GM-CSF are increased locally and insome cases systemically and have been correlated with the inflammatoryprocess in these diseases.

It is therefore an aim of the invention to improve on the modes ofneutralizing increased and/or undesired GM-CSF activity previously knownin the prior art.

Accordingly one aspect of the invention relates to a human monoclonalantibody or fragment thereof which specifically binds to and neutralizesprimate GM-CSF.

The term “specifically binds” or related expressions such as “specificbinding”, “binding specifically”, “specific binder” etc. as used hereinrefer to the ability of the human monoclonal antibody or fragmentthereof to discriminate between primate GM-CSF and any number of otherpotential antigens different from primate GM-CSF to such an extent that,from a pool of a plurality of different antigens as potential bindingpartners, only primate GM-CSF is bound, or is significantly bound.Within the meaning of the invention, primate GM-CSF is “significantly”bound when, from among a pool of a plurality of equally accessibledifferent antigens as potential binding partners, primate GM-CSF isbound at least 10-fold, preferably 50-fold, most preferably 100-fold orgreater more frequently (in a kinetic sense) than any other antigendifferent than primate GM-CSF. Such kinetic measurements can beperformed on a Biacore apparatus.

As used herein, “neutralization,” “neutralizer,” “neutralizing” andgrammatically related variants thereof refer to partial or completeattenuation of the biological effect(s) of GM-CSF. Such partial orcomplete attenuation of the biological effect(s) of GM-CSF results frommodification, interruption and/or abrogation of GM-CSF-mediated signaltransduction, as manifested, for example, in intracellular signalling,cellular proliferation or release of soluble substances, up- ordown-regulation of intracellular gene activation, for example thatresulting in expression of surface receptors for ligands other thanGM-CSF. As one of skill in the art understands, there exist multiplemodes of determining whether an agent, for example an antibody inquestion or fragment thereof is to be classified as a neutralizer. As anexample, this may be accomplished by a standard in vitro test performedgenerally as follows: In a first proliferation experiment, a cell line,the degree of proliferation of which is known to depend on the activityof GM-CSF, is incubated in a series of samples with varyingconcentrations of GM-CSF, following which incubation the degree ofproliferation of the cell line is measured. From this measurement, theconcentration of GM-CSF allowing half-maximal proliferation of the cellsis determined A second proliferation experiment is then performedemploying in each of a series of samples the same number of cells asused in the first proliferation experiment, the above-determinedconcentration of GM-CSF and, this time, varying concentrations of anantibody or fragment thereof suspected of being a neutralizer of GM-CSF.Cell proliferation is again measured to determine the concentration ofantibody or fragment thereof sufficient to effect half-maximal growthinhibition. If the resulting graph of growth inhibition vs.concentration of antibody (or fragment thereof) is sigmoidal in shape,resulting in decreased cell proliferation with increasing concentrationof antibody (or fragment thereof), then some degree ofantibody-dependent growth inhibition has been effected, i.e. theactivity of GM-CSF has been neutralized to some extent. In such a case,the antibody or fragment thereof may be considered a “neutralizer” inthe sense of the present invention. One example of a cell line, thedegree of proliferation of which is known to depend on the activity ofGM-CSF, is the TF-1 cell line, as described in Kitamura, T. et al.(1989). J Cell Physiol 140, 323-34.

As one of ordinary skill in the art understands, the degree of cellularproliferation is not the only parameter by which neutralizing capacitymay be established. For example, measurement of the level of signallingmolecules (e.g. cytokines), the level of secretion of which depends onGM-CSF, may be used to identify a suspected GM-CSF neutralizer.

Other examples of cell lines which can be used to determine whether anantibody in question or fragment thereof is a neutralizer of primateGM-CSF activity include AML-193 (Lange, B. et al. (1987). Blood 70,192-9); GF-D8 (Rambaldi, A. et al. (1993). Blood 81, 1376-83); GM/SO(Oez, S. et al. (1990). Experimental Hematology 18, 1108-11); MO7E(Avanzi, G. C. et al. (1990). Journal of Cellular Physiology 145,458-64); TALL-103 (Valtieri, M. et al. (1987). Journal of Immunology138, 4042-50); UT-7 (Komatsu, N. et al. (1991). Cancer Research 51,341-8).

The human antibody or fragment thereof according to the invention ismonoclonal. As used herein, the term “monoclonal” is to be understood ashaving the meaning typically ascribed to it in the art, namely anantibody (or its corresponding fragment) arising from a single clone ofan antibody-producing cell such as a B cell, and recognizing a singleepitope on the antigen bound. It is particularly difficult to preparehuman antibodies which are monoclonal. In contrast to fusions of murineB cells with immortalized cell lines, fusions of human B cells withimmortalized cell lines are not viable. Thus, the human monoclonalantibody of the invention is the result of overcoming significanttechnical hurdles generally acknowledged to exist in the field ofantibody technology. The monoclonal nature of the antibody makes itparticularly well suited for use as a therapeutic agent, since suchantibody will exist as a single, homogeneous molecular species which canbe well-characterized and reproducibly made and purified. These factorsresult in a product whose biological activity can be predicted with ahigh level of precision, very important if such a molecule is going togain regulatory approval for therapeutic administration in humans.

It is especially important that the monoclonal antibody (orcorresponding fragment) according to the invention be a human antibody(or corresponding fragment). In contemplating an antibody agent intendedfor therapeutic administration to humans, it is highly advantageous thatthis antibody is of human origin. Following administration to a humanpatient, a human antibody or fragment thereof will most probably notelicit a strong immunogenic response by the patient's immune system,i.e. will not be recognized as being a “foreign”, that is non-humanprotein. This means that no host, i.e. patient antibodies will begenerated against the therapeutic antibody which would otherwise blockthe therapeutic antibody's activity and/or accelerate the therapeuticantibody's elimination from the body of the patient, thus preventing itfrom exerting its desired therapeutic effect.

The term “human” antibody as used herein is to be understood as meaningthat the antibody of the invention, or its feagment, comprises (an)amino acid sequence(s) contained in the human germline antibodyrepertoire. For the purposes of definition herein, an antibody, or itsfeagment, may therefore be considered human if it consists of such (a)human germline amino acid sequence(s), i.e. if the amino acidsequence(s) of the antibody in question or fragment thereof is (are)identical to (an) expressed human germline amino acid sequence(s). Anantibody or fragment thereof may also be regarded as human if itconsists of (a) sequence(s) that deviate(s) from its (their) closesthuman germline sequence(s) by no more than would be expected due to theimprint of somatic hypermutation. Additionally, the antibodies of manynon-human mammals, for example rodents such as mice and rats, compriseVH CDR3 amino acid sequences which one may expect to exist in theexpressed human antibody repertoire as well. Any such sequence(s) ofhuman or non-human origin which may be expected to exist in theexpressed human repertoire would also be considered “human” for thepurposes of the present invention.

According to one embodiment of the invention, the primate GM-CSF ishuman (Homo sapiens) GM-CSF or non-human primate GM-CSF. Especiallypreferred variants of non-human primate GM-CSF include gibbon monkey(nomascus concolor, also known as the western black crested gibbon)GM-CSF and GM-CSF of monkeys of the macaca family, for example rhesusmonkey (Macaca mulatta) GM-CSF and cynomolgous monkey (Macacafascicularis) GM-CSF. According to this embodiment of the invention, thehuman monoclonal antibody or fragment thereof exhibits cross reactivitybetween both human and at least one of the monkey species mentionedabove. This is especially advantageous for an antibody molecule which isintended for therapeutic administration in human subjects, since such anantibody will normally have to proceed through a multitude of testsprior to regulatory approval, of which certain early tests involvenon-human animal species. In performing such tests, it is generallydesirable to use as a non-human species a species bearing a high degreeof genetic similarity to humans, since the results so obtained willgenerally be highly predictive of corresponding results which may beexpected when administering the same molecule to humans. However, suchpredictive power based on animal tests depends at least partially on thecomparability of the molecule, and is very high when, due to across-species reactivity, the same therapeutic molecule may beadministered to humans and animal models. As in this embodiment of theinvention, when an antibody molecule is cross reactive for the sameantigen in humans as in another closely related species, tests may beperformed using the same antibody molecule in humans as in this closelyrelated species, for example in one of the monkey species mentionedabove. This increases both the efficiency of the tests themselves aswell as predictive power allowed by such tests regarding the behavior ofsuch antibodies in humans, the ultimate species of interest from atherapeutic standpoint.

According to a further embodiment of the invention, the human monoclonalantibody may be an IgG antibody. As is well known in the art, an IgGcomprises not only the variable antibody regions responsible for thehighly discriminative antigen recognition and binding, but also theconstant regions of the heavy and light antibody polypeptide chainsnormally present in endogenously produced antibodies and, in some cases,even decoration at one or more sites with carbohydrates. Suchglycosylation is generally a hallmark of the IgG format, and portions ofthese constant regions make up the so called Fc region of a fullantibody which is known to elicit various effector functions in vivo. Inaddition, the Fc region mediates binding of IgG to Fc receptor, henceprolonging half life in vivo as well as facilitating homing of the IgGto locations with increased Fc receptor presence—inflamed tissue, forexample. Advantageously, the IgG antibody is an IgG1 antibody or an IgG4antibody, formats which are preferred since their mechanism of action invivo is particularly well understood and characterized. This isespecially the case for IgG1 antibodies.

According to a further embodiment of the invention, the fragment of thehuman monoclonal antibody may be an scFv, a single domain antibody, anFv, a VHH antibody, a diabody, a tandem diabody, a Fab, a Fab' or aF(ab)2. These formats may generally be divided into two subclasses,namely those which consist of a single polypeptide chain, and thosewhich comprise at least two polypeptide chains. Members of the formersubclass include an scFv (comprising one VH region and one VL regionjoined into a single polypeptide chain via a polypeptide linker); asingle domain antibody (comprising a single antibody variable region)such as a VHH antibody (comprising a single VH region). Members of thelatter subclass include an Fv (comprising one VH region and one VLregion as separate polypeptide chains which are non-covalentlyassociated with one another); a diabody (comprising two non-covalentlyassociated polypeptide chains, each of which comprises two antibodyvariable regions—normally one VH and one VL per polypeptide chain—thetwo polypeptide chains being arranged in a head-to-tail conformation sothat a bivalent antibody molecule results); a tandem diabody (bispecificsingle-chain Fv antibodies comprising four covalently linkedimmunoglobulin variable—VH and VL—regions of two differentspecificities, forming a homodimer that is twice as large as the diabodydescribed above); a Fab (comprising as one polypeptide chain an entireantibody light chain, itself comprising a VL region and the entire lightchain constant region and, as another polypeptide chain, a part of anantibody heavy chain comprising a complete VH region and part of theheavy chain constant region, said two polypeptide chains beingintermolecularly connected via an interchain disulfide bond); a Fab' (asa Fab, above, except with additional reduced disulfide bonds comprisedon the antibody heavy chain); and a F(ab)2 (comprising two Fab'molecules, each Fab' molecule being linked to the respective other Fab'molecule via interchain disulfide bonds). In general, antibody fragmentsof the type described hereinabove allow great flexibility in tailoring,for example, the pharmacokinetic properties of an antibody desired fortherapeutic administration to the particular exigencies at hand. Forexample, it may be desirable to reduce the size of the antibodyadministered in order to increase the degree of tissue penetration whentreating tissues known to be poorly vascularized (for example, joints).Under some circumstances, it may also be desirable to increase the rateat which the therapeutic antibody is eliminated from the body, said rategenerally being acceleratable by decreasing the size of the antibodyadministered.

According to a further embodiment of the invention, said humanmonoclonal antibody or fragment thereof may be present in monovalentmonospecific; multivalent monospecific, in particular bivalentmonospecific; or multivalent multispecific, in particular bivalentbispecific forms. In general, a multivalent monospecific, in particularbivalent monospecific antibody such as a full human IgG as describedhereinabove may bring with it the therapeutic advantage that theneutralization effected by such an antibody is potentiated by avidityeffects, i.e. binding by the same antibody to multiple molecules of thesame antigen, here primate GM-CSF. Several monovalent monospecific formsof fragments of the antibody of the invention have been described above(for example, an scFv, an Fv, a VHH or a single domain antibody).Multivalent multispecific, in particular bivalent bispecific forms ofthe human monoclonal anti-primate GM-CSF antibody of the invention mayinclude a full IgG in which one binding arm binds to primate GM-CSFwhile the other binding arm of which binds to another antigen differentfrom primate GM-CSF. A further multivalent multispecific, in particularbivalent bispecific form may advantageously be a human single chainbispecific antibody, i.e. a recombinant human antibody constructcomprising two scFv entities as described above, connected into onecontiguous polypeptide chain by a short interposed polypeptide spacer asgenerally known in the art (see for example WO 99/54440 for ananti-CD19×anti-CD3 bispecific single chain antibody). Here, one scFvportion of the bispecific single chain antibody comprised within thebispecific single chain antibody will specifically bind primate GM-CSFas set out above, while the respective other scFv portion of thisbispecific single chain antibody will bind another antigen determined tobe of therapeutic benefit.

According to a further embodiment the human monoclonal antibody orfragment thereof may be derivatized, for example with an organicpolymer, for example with one or more molecules of polyethylene glycol(“PEG”) and/or polyvinyl pyrrolidone (“PVP”). As is known in the art,such derivatization can be advantageous in modulating thepharmacodynamic properties of antibodies or fragments thereof.Especially preferred are PEG molecules derivatized as PEG-maleimide,enabling conjugation with the antibody or fragment thereof in asite-specific manner via the sulfhydryl group of a cysteine amino acid.Of these, especially preferred are 20kD and/or 40 kD PEG-maleimide, ineither branched or straight-chain form. It may be especiallyadvantageous to increase the effective molecular weight of smaller humananti-primate GM-CSF antibody fragments such as scFv fragments bycoupling the latter to one or more molecules of PEG, especiallyPEG-maleimide.

According to a further embodiment of the invention, the human monoclonalantibody or fragment thereof specifically binds to an epitope, inparticular to a discontinuous epitope, of human or non-human primateGM-CSF comprising amino acids 23-27 (RRLLN) and/or amino acids 65-77(GLR/QGSLTKLKGPL).

The variability at position 67 within the amino acid sequence stretch65-77 depicted above reflects the heterogeneity in this portion ofprimate GM-CSF between, on the one hand, human and gibbon GM-CSF (inwhich position 67 is R) and, on the other hand, monkeys of the macacafamily, for example cynomolgous and rhesus monkeys (in which position 67is Q).

As used herein, the numbering of human and non-human primate GM-CSFrefers to that of mature GM-CSF, i.e. GM-CSF without its 17 amino acidsignal sequence (the total length of mature GM-CSF in both human andnon-human primate species described above is 127 amino acids). Thesequence of human GM-CSF and gibbon GM-CSF is as follows:

APARSPSPST QPWEHVNAIQ EA RRLLN LSR D TAAEMNETVEVISEMFDLQ EPTCLQTRLE LYKQGLRGSL TKLKGPLTMMASHYKQHCPP TPETSCATQI ITFESFKENL KDFLLVIPFD CWEPVQE

The sequence of GM-CSF in certain members of the macaca monkey familysuch as for example rhesus monkey and cynomolgous monkey is as follows:

APARSPSPGT QPWEHVNAIQ EA RRLLN LSR D TAAEMNKTVEVVSEMFDLQ EPSCLQTRLE LYKQGLQGSL TKLKGPLTMMASHYKQHCPP TPETSCATQI ITFQSFKENL KDFLLVIPFD CWEPVQE

The minimum epitope, advantageously a discontinuous epitope, bound bythe human monoclonal antibody of the invention (or fragment thereof) asdescribed above is indicated in the above GM-CSF sequence in boldface.As used herein, the term “discontinuous epitope” is to be understood asat least two non-adjacent amino acid sequence stretches within a givenpolypeptide chain, here mature human and non-human primate GM-CSF, whichare simultaneously and specifically (as defined above) bound by anantibody. According to this definition, such simultaneous specificbinding may be of the GM-CSF polypeptide in linear form. Here, one mayimagine the mature GM-CSF polypeptide forming an extended loop, in oneregion of which the two sequences indicated in boldface above line up,for example more or less in parallel and in proximity of one another. Inthis state they are specifically and simultaneously bound by theantibody fragment of the invention. According to this definition,simultaneous specific binding of the two sequence stretches of matureGM-CSF indicated above may also take the form of antibody binding to aconformational epitope. Here, mature GM-CSF has already formed itstertiary conformation as it normally exists in vivo (Sun, H. W., J.Bernhagen, et al. (1996). Proc Natl Acad Sci USA 93, 5191-6). In thistertiary conformation, the polypeptide chain of mature GM-CSF is foldedin such a manner as to bring the two sequence stretches indicated aboveinto spatial proximity, for example on the outer surface of a particularregion of mature, folded GM-CSF, where they are then recognized byvirtue of their three-dimensional conformation in the context of thesurrounding polypeptide sequences.

In a preferred embodiment, the above (discontinuous) epitope furthercomprises amino acids 28-31 (LSRD), italicized in the above sequences ofhuman and non-human primate GM-CSF. In an especially preferredembodiment, either of the above (discontinuous) epitopes furthercomprises amino acids 32-33 (TA) and/or amino acids 21-22 (EA), each ofwhich stretch is underlined in the above sequences of human andnon-human primate GM-CSF.

According to a further embodiment of the invention, the human monoclonalantibody or fragment thereof comprises in its heavy chain variableregion a CDR3 comprising an amino acid sequence chosen from the groupconsisting of those set out in any of the SEQ ID NOs: 1-13 or 56.Preferred is a human monoclonal antibody or fragment thereof comprisinga heavy chain variable region CDR1 sequence as set out in SEQ ID NO: 14,a heavy chain variable region CDR2 sequence as set out in SEQ ID NO: 15and a heavy chain variable region CDR3 sequence as set out in SEQ ID NO:1; or comprising a heavy chain variable region CDR1 sequence as set outin SEQ ID NO: 14, a heavy chain variable region CDR2 sequence as set outin SEQ ID NO: 15 and a heavy chain variable region CDR3 sequence as setout in SEQ ID NO: 2; or comprising a heavy chain variable region CDR1sequence as set out in SEQ ID NO: 14, a heavy chain variable region CDR2sequence as set out in SEQ ID NO: 15 and a heavy chain variable regionCDR3 sequence as set out in SEQ ID NO: 3; or comprising a heavy chainvariable region CDR1 sequence as set out in SEQ ID NO: 14, a heavy chainvariable region CDR2 sequence as set out in SEQ ID NO: 15 and a heavychain variable region CDR3 sequence as set out in SEQ ID NO: 4; orcomprising a heavy chain variable region CDR1 sequence as set out in SEQID NO: 14, a heavy chain variable region CDR2 sequence as set out in SEQID NO: 15 and a heavy chain variable region CDR3 sequence as set out inSEQ ID NO: 5; or comprising a heavy chain variable region CDR1 sequenceas set out in SEQ ID NO: 14, a heavy chain variable region CDR2 sequenceas set out in SEQ ID NO: 15 and a heavy chain variable region CDR3sequence as set out in SEQ ID NO: 6; or comprising a heavy chainvariable region CDR1 sequence as set out in SEQ ID NO: 14, a heavy chainvariable region CDR2 sequence as set out in SEQ ID NO: 15 and a heavychain variable region CDR3 sequence as set out in SEQ ID NO: 7; orcomprising a heavy chain variable region CDR1 sequence as set out in SEQID NO: 14, a heavy chain variable region CDR2 sequence as set out in SEQID NO: 15 and a heavy chain variable region CDR3 sequence as set out inSEQ ID NO: 8; or comprising a heavy chain variable region CDR1 sequenceas set out in SEQ ID NO: 14, a heavy chain variable region CDR2 sequenceas set out in SEQ ID NO: 15 and a heavy chain variable region CDR3sequence as set out in SEQ ID NO: 9; or comprising a heavy chainvariable region CDR1 sequence as set out in SEQ ID NO: 14, a heavy chainvariable region CDR2 sequence as set out in SEQ ID NO: 15 and a heavychain variable region CDR3 sequence as set out in SEQ ID NO: 10; orcomprising a heavy chain variable region CDR1 sequence as set out in SEQID NO: 14, a heavy chain variable region CDR2 sequence as set out in SEQID NO: 15 and a heavy chain variable region CDR3 sequence as set out inSEQ ID NO: 11; or comprising a heavy chain variable region CDR1 sequenceas set out in SEQ ID NO: 14, a heavy chain variable region CDR2 sequenceas set out in SEQ ID NO: 15 and a heavy chain variable region CDR3sequence as set out in SEQ ID NO: 12; or comprising a heavy chainvariable region CDR1 sequence as set out in SEQ ID NO: 14, a heavy chainvariable region CDR2 sequence as set out in SEQ ID NO: 15 and a heavychain variable region CDR3 sequence as set out in SEQ ID NO: 13; orcomprising a heavy chain variable region CDR1 sequence as set out in SEQID NO: 14, a heavy chain variable region CDR2 sequence as set out in SEQID NO: 15 and a heavy chain variable region CDR3 sequence as set out inSEQ ID NO: 56.

Still more preferred, any of the above 14 combinations of CDR1, CDR2 andCDR3 sequences exists in a human monoclonal antibody or fragment thereoffurther comprising in its light chain variable region a CDR1 comprisingthe amino acid sequence set out in SEQ ID NO: 16, a CDR2 comprising theamino acid sequence set out in SEQ ID NO: 17, and a CDR3 comprising theamino acid sequence set out in SEQ ID NO: 18.

According to a further embodiment, the human monoclonal antibody of theinvention or fragment thereof comprises in its light chain variableregion an amino acid sequence as set out in SEQ ID NO. 19. Preferred isa human monoclonal antibody or fragment thereof, the light chainvariable region comprising an amino acid sequence as set out in SEQ IDNO. 19 and a heavy chain variable region comprising an amino acidsequence as set out in SEQ ID NO: 20; or a human monoclonal antibody orfragment thereof, the light chain variable region comprising an aminoacid sequence as set out in SEQ ID NO. 19 and a heavy chain variableregion comprising an amino acid sequence as set out in SEQ ID NO: 21; ora human monoclonal antibody or fragment thereof, the light chainvariable region comprising an amino acid sequence as set out in SEQ IDNO. 19 and a heavy chain variable region comprising an amino acidsequence as set out in SEQ ID NO: 22; or a human monoclonal antibody orfragment thereof, the light chain variable region comprising an aminoacid sequence as set out in SEQ ID NO. 19 and a heavy chain variableregion comprising an amino acid sequence as set out in SEQ ID NO: 23; ora human monoclonal antibody or fragment thereof, the light chainvariable region comprising an amino acid sequence as set out in SEQ IDNO. 19 and a heavy chain variable region comprising an amino acidsequence as set out in SEQ ID NO: 24; or a human monoclonal antibody orfragment thereof, the light chain variable region comprising an aminoacid sequence as set out in SEQ ID NO. 19 and a heavy chain variableregion comprising an amino acid sequence as set out in SEQ ID NO: 25; ora human monoclonal antibody or fragment thereof, the light chainvariable region comprising an amino acid sequence as set out in SEQ IDNO. 19 and a heavy chain variable region comprising an amino acidsequence as set out in SEQ ID NO: 26; or a human monoclonal antibody orfragment thereof, the light chain variable region comprising an aminoacid sequence as set out in SEQ ID NO. 19 and a heavy chain variableregion comprising an amino acid sequence as set out in SEQ ID NO: 27; ora human monoclonal antibody or fragment thereof, the light chainvariable region comprising an amino acid sequence as set out in SEQ IDNO. 19 and a heavy chain variable region comprising an amino acidsequence as set out in SEQ ID NO: 28; or a human monoclonal antibody orfragment thereof, the light chain variable region comprising an aminoacid sequence as set out in SEQ ID NO. 19 and a heavy chain variableregion comprising an amino acid sequence as set out in SEQ ID NO: 29; ora human monoclonal antibody or fragment thereof, the light chainvariable region comprising an amino acid sequence as set out in SEQ IDNO. 19 and a heavy chain variable region comprising an amino acidsequence as set out in SEQ ID NO: 30; or a human monoclonal antibody orfragment thereof, the light chain variable region comprising an aminoacid sequence as set out in SEQ ID NO. 19 and a heavy chain variableregion comprising an amino acid sequence as set out in SEQ ID NO: 31; ora human monoclonal antibody or fragment thereof, the light chainvariable region comprising an amino acid sequence as set out in SEQ IDNO. 19 and a heavy chain variable region comprising an amino acidsequence as set out in SEQ ID NO: 32; or a human monoclonal antibody orfragment thereof, the light chain variable region comprising an aminoacid sequence as set out in SEQ ID NO. 19 and a heavy chain variableregion comprising an amino acid sequence as set out in SEQ ID NO: 33; ora human monoclonal antibody or fragment thereof, the light chainvariable region comprising an amino acid sequence as set out in SEQ IDNO. 19 and a heavy chain variable region comprising an amino acidsequence as set out in SEQ ID NO: 52; or a human monoclonal antibody orfragment thereof, the light chain variable region comprising an aminoacid sequence as set out in SEQ ID NO. 19 and a heavy chain variableregion comprising an amino acid sequence as set out in SEQ ID NO: 53.

According to a further embodiment, the human monoclonal antibody of theinvention or fragment thereof comprises in its light chain variableregion an amino acid sequence as set out in SEQ ID NO. 54. Preferred isa human monoclonal antibody or fragment thereof, the light chainvariable region comprising an amino acid sequence as set out in SEQ IDNO. 54 and a heavy chain variable region comprising an amino acidsequence as set out in SEQ ID NO: 20; or a human monoclonal antibody orfragment thereof, the light chain variable region comprising an aminoacid sequence as set out in SEQ ID NO. 54 and a heavy chain variableregion comprising an amino acid sequence as set out in SEQ ID NO: 21; ora human monoclonal antibody or fragment thereof, the light chainvariable region comprising an amino acid sequence as set out in SEQ IDNO. 54 and a heavy chain variable region comprising an amino acidsequence as set out in SEQ ID NO: 22; or a human monoclonal antibody orfragment thereof, the light chain variable region comprising an aminoacid sequence as set out in SEQ ID NO. 54 and a heavy chain variableregion comprising an amino acid sequence as set out in SEQ ID NO: 23; ora human monoclonal antibody or fragment thereof, the light chainvariable region comprising an amino acid sequence as set out in SEQ IDNO. 54 and a heavy chain variable region comprising an amino acidsequence as set out in SEQ ID NO: 24; or a human monoclonal antibody orfragment thereof, the light chain variable region comprising an aminoacid sequence as set out in SEQ ID NO. 54 and a heavy chain variableregion comprising an amino acid sequence as set out in SEQ ID NO: 25; ora human monoclonal antibody or fragment thereof, the light chainvariable region comprising an amino acid sequence as set out in SEQ IDNO. 54 and a heavy chain variable region comprising an amino acidsequence as set out in SEQ ID NO: 26; or a human monoclonal antibody orfragment thereof, the light chain variable region comprising an aminoacid sequence as set out in SEQ ID NO. 54 and a heavy chain variableregion comprising an amino acid sequence as set out in SEQ ID NO: 27; ora human monoclonal antibody or fragment thereof, the light chainvariable region comprising an amino acid sequence as set out in SEQ IDNO. 54 and a heavy chain variable region comprising an amino acidsequence as set out in SEQ ID NO: 28; or a human monoclonal antibody orfragment thereof, the light chain variable region comprising an aminoacid sequence as set out in SEQ ID NO. 54 and a heavy chain variableregion comprising an amino acid sequence as set out in SEQ ID NO: 29; ora human monoclonal antibody or fragment thereof, the light chainvariable region comprising an amino acid sequence as set out in SEQ IDNO. 54 and a heavy chain variable region comprising an amino acidsequence as set out in SEQ ID NO: 30; or a human monoclonal antibody orfragment thereof, the light chain variable region comprising an aminoacid sequence as set out in SEQ ID NO. 54 and a heavy chain variableregion comprising an amino acid sequence as set out in SEQ ID NO: 31; ora human monoclonal antibody or fragment thereof, the light chainvariable region comprising an amino acid sequence as set out in SEQ IDNO. 54 and a heavy chain variable region comprising an amino acidsequence as set out in SEQ ID NO: 32; or a human monoclonal antibody orfragment thereof, the light chain variable region comprising an aminoacid sequence as set out in SEQ ID NO. 54 and a heavy chain variableregion comprising an amino acid sequence as set out in SEQ ID NO: 33; ora human monoclonal antibody or fragment thereof, the light chainvariable region comprising an amino acid sequence as set out in SEQ IDNO. 54 and a heavy chain variable region comprising an amino acidsequence as set out in SEQ ID NO: 52; or a human monoclonal antibody orfragment thereof, the light chain variable region comprising an aminoacid sequence as set out in SEQ ID NO. 54 and a heavy chain variableregion comprising an amino acid sequence as set out in SEQ ID NO: 53.

According to a further embodiment, the human monoclonal antibody of theinvention or fragment thereof comprises in its light chain variableregion an amino acid sequence as set out in SEQ ID NO. 55. Preferred isa human monoclonal antibody or fragment thereof, the light chainvariable region comprising an amino acid sequence as set out in SEQ IDNO. 55 and a heavy chain variable region comprising an amino acidsequence as set out in SEQ ID NO: 20; or a human monoclonal antibody orfragment thereof, the light chain variable region comprising an aminoacid sequence as set out in SEQ ID NO. 55 and a heavy chain variableregion comprising an amino acid sequence as set out in SEQ ID NO: 21; ora human monoclonal antibody or fragment thereof, the light chainvariable region comprising an amino acid sequence as set out in SEQ IDNO. 55 and a heavy chain variable region comprising an amino acidsequence as set out in SEQ ID NO: 22; or a human monoclonal antibody orfragment thereof, the light chain variable region comprising an aminoacid sequence as set out in SEQ ID NO. 55 and a heavy chain variableregion comprising an amino acid sequence as set out in SEQ ID NO: 23; ora human monoclonal antibody or fragment thereof, the light chainvariable region comprising an amino acid sequence as set out in SEQ IDNO. 55 and a heavy chain variable region comprising an amino acidsequence as set out in SEQ ID NO: 24; or a human monoclonal antibody orfragment thereof, the light chain variable region comprising an aminoacid sequence as set out in SEQ ID NO. 55 and a heavy chain variableregion comprising an amino acid sequence as set out in SEQ ID NO: 25; ora human monoclonal antibody or fragment thereof, the light chainvariable region comprising an amino acid sequence as set out in SEQ IDNO. 55 and a heavy chain variable region comprising an amino acidsequence as set out in SEQ ID NO: 26; or a human monoclonal antibody orfragment thereof, the light chain variable region comprising an aminoacid sequence as set out in SEQ ID NO. 55 and a heavy chain variableregion comprising an amino acid sequence as set out in SEQ ID NO: 27; ora human monoclonal antibody or fragment thereof, the light chainvariable region comprising an amino acid sequence as set out in SEQ IDNO. 55 and a heavy chain variable region comprising an amino acidsequence as set out in SEQ ID NO: 28; or a human monoclonal antibody orfragment thereof, the light chain variable region comprising an aminoacid sequence as set out in SEQ ID NO. 55 and a heavy chain variableregion comprising an amino acid sequence as set out in SEQ ID NO: 29; ora human monoclonal antibody or fragment thereof, the light chainvariable region comprising an amino acid sequence as set out in SEQ IDNO. 55 and a heavy chain variable region comprising an amino acidsequence as set out in SEQ ID NO: 30; or a human monoclonal antibody orfragment thereof, the light chain variable region comprising an aminoacid sequence as set out in SEQ ID NO. 55 and a heavy chain variableregion comprising an amino acid sequence as set out in SEQ ID NO: 31; ora human monoclonal antibody or fragment thereof, the light chainvariable region comprising an amino acid sequence as set out in SEQ IDNO. 55 and a heavy chain variable region comprising an amino acidsequence as set out in SEQ ID NO: 32; or a human monoclonal antibody orfragment thereof, the light chain variable region comprising an aminoacid sequence as set out in SEQ ID NO. 55 and a heavy chain variableregion comprising an amino acid sequence as set out in SEQ ID NO: 33; ora human monoclonal antibody or fragment thereof, the light chainvariable region comprising an amino acid sequence as set out in SEQ IDNO. 55 and a heavy chain variable region comprising an amino acidsequence as set out in SEQ ID NO: 52; or a human monoclonal antibody orfragment thereof, the light chain variable region comprising an aminoacid sequence as set out in SEQ ID NO. 55 and a heavy chain variableregion comprising an amino acid sequence as set out in SEQ ID NO: 53.

A preferred embodiment provides a human monoclonal antibody or fragmentthereof comprising in its light chain a variable region a CDR1 regioncomprising an amino acid sequence as set out in SEQ ID NO. 16, a CDR2region having an amino acid sequence as set out in SEQ ID NO. 17 and aCDR3 having an amino acid sequence as set out in SEQ ID NO. 18 andcomprising in its heavy chain variable region a CDR1 region comprisingan amino acid sequence as set out in SEQ ID NO. 14, a CDR2 region havingan amino acid sequence as set out in SEQ ID NO. 15 and a CDR3 having anamino acid sequence as set out in any of SEQ ID NOs. 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13 or 56.

In a further preferred embodiment the human monoclonal antibodycomprises in its light chain an amino acid sequence as set out in SEQ IDNO: 34 and in its heavy chain an amino acid sequence as set out in SEQID NO: 35; or in its light chain an amino acid sequence as set out inSEQ ID NO: 34 and in its heavy chain an amino acid sequence as set outin SEQ ID NO: 36; or in its light chain an amino acid sequence as setout in SEQ ID NO: 34 and in its heavy chain an amino acid sequence asset out in SEQ ID NO: 37; or in its light chain an amino acid sequenceas set out in SEQ ID NO: 34 and in its heavy chain an amino acidsequence as set out in SEQ ID NO: 38; or in its light chain an aminoacid sequence as set out in SEQ ID NO: 34 and in its heavy chain anamino acid sequence as set out in SEQ ID NO: 39; or in its light chainan amino acid sequence as set out in SEQ ID NO: 34 and in its heavychain an amino acid sequence as set out in SEQ ID NO: 40; or in itslight chain an amino acid sequence as set out in SEQ ID NO: 34 and inits heavy chain an amino acid sequence as set out in SEQ ID NO: 41; orin its light chain an amino acid sequence as set out in SEQ ID NO: 34and in its heavy chain an amino acid sequence as set out in SEQ ID NO:42; or in its light chain an amino acid sequence as set out in SEQ IDNO: 34 and in its heavy chain an amino acid sequence as set out in SEQID NO: 43; or in its light chain an amino acid sequence as set out inSEQ ID NO: 34 and in its heavy chain an amino acid sequence as set outin SEQ ID NO: 44; or in its light chain an amino acid sequence as setout in SEQ ID NO: 34 and in its heavy chain an amino acid sequence asset out in SEQ ID NO: 45; or in its light chain an amino acid sequenceas set out in SEQ ID NO: 34 and in its heavy chain an amino acidsequence as set out in SEQ ID NO: 46; or in its light chain an aminoacid sequence as set out in SEQ ID NO: 34 and in its heavy chain anamino acid sequence as set out in SEQ ID NO: 47; or in its light chainan amino acid sequence as set out in SEQ ID NO: 34 and in its heavychain an amino acid sequence as set out in SEQ ID NO: 48.

The preferred embodiments above provide human monoclonal antibodymolecules and/or fragments thereof which are especially advantageous asneutralizers of the activity of primate, especially of human GM-CSF.Human monoclonal antibodies or fragments thereof according to theseespecially preferred embodiments are highly advantageous for severalreasons.

First, they recognize primate GM-CSF highly specifically, that is to saythat from a mixture of primate GM-CSF with other primate colonystimulating factors (for example primate G-CSF and M-CSF), the bindingmolecules according to these especially preferred embodiments are highlydiscriminating for primate GM-CSF, whereas the other colony stimulatingfactors in the same milieu are not recognized. This means that a humanmonoclonal antibody or fragment thereof according to these embodiments,when administered to a human, will be expected to specifically bind toand neutralize only the desired target, whereas other undesired targetsare neither bound nor neutralized. Ultimately, this leads to a highdegree of predictability concerning the therapeutic mode of action invivo.

Second, binders according to these especially preferred embodiments bindto primate GM-CSF with extremely high affinity. K_(D) values of fromabout 4×10⁻⁹ M down to as low as about 0.04×10⁻⁹ M, the lattercorresponding to about 40 pM, have been observed for molecules of thisclass. Since the kinetic on-rate of such molecules in aqueous media islargely diffusion controlled and therefore cannot be improved beyondwhat the local diffusion conditions will allow under physiologicalconditions, the low K_(D) arises primarily as a result of the kineticoff-rate, k_(off), which for the highest affinity antibody binder isapproximately 10⁻⁵ s⁻¹. This means that once the complex between a humanmonoclonal antibody or fragment thereof according to any of theseembodiments of the invention on the one hand and primate GM-CSF on theother hand is formed, it does not readily, or at least does not quicklyseparate. For binding molecules intended as neutralizers of biologicalactivity, these characteristics are highly advantageous since thedesirable neutralizing effect will normally last only as long as themolecule, the biological activity of which is to be neutralized (hereprimate GM-CSF) remains bound by the neutralizing binding molecule. So aneutralizing molecule which remains bound to its intended target for along time will continue to neutralize for a correspondingly long time.

The high binding affinity of human monoclonal antibodies or fragmentsthereof to primate GM-CSF has an additional advantage. Normally,antibodies or fragments thereof will be eliminated from the bloodstreamof a patient in a size-dependent fashion, with smaller molecules beingexcreted and eliminated before larger ones. Since the complex of the twopolypeptides—antibody or antibody fragment and bound GM-CSF—is obviouslylarger than the antibody alone, the low k_(off) mentioned above has theeffect that therapeutic neutralizer is excreted and eliminated from thepatient's body more slowly than would be the case, were it not bound toGM-CSF. Thus, not only the magnitude of the neutralizing activity butalso its duration in vivo is increased.

Finally, the neutralizing activity determined for binders according tothese especially preferred embodiments is surprisingly high. As will bedescribed in more detail herein below, neutralizing activity wasmeasured in vitro using a TF-1 growth inhibition assay (Kitamura, T. etal. (1989). J Cell Physiol 140, 323-34). As an indication ofneutralizing potential, IC₅₀ values were measured, IC₅₀ representing theconcentration of human monoclonal antibody or fragment thereof accordingto any of these embodiments of the invention required to elicit ahalf-maximal inhibition of TF-1 cell proliferation. For human monoclonalantibodies or fragments thereof according to any of these embodiments ofthe invention an IC₅₀ value of approximately 3×10⁻¹⁰ M, or about 0.3 nMwas determined The binding molecules according to any of theseembodiments of the invention are therefore highly potent neutralizers ofthe activity of primate GM-CSF.

In summary, then, a human monoclonal antibody or fragment thereofaccording to any of the above embodiments of the invention exhibits highdegree of discrimination for the desired antigen, binds this antigenextremely tightly and for a long time and exhibits highly potentneutralizing activity for the long time it remains bound. At the sametime, the long persistence of the binder-antigen complex slowselimination of this binder from the body, thereby lengthening theduration of the desired therapeutic effect in vivo.

A further aspect of the invention provides a human monoclonal antibodyor fragment thereof comprising an amino acid sequence having at least70% homology with an amino acid as set out in any of SEQ ID NOs: 1-48and/or 52-56. Homology may be determined by standard sequence alignmentprograms such as Vector NTI (InforMax™, Maryland, USA). Such programscompare aligned sequences on an amino acid-by-amino acid basis, and canbe set to various levels of stringency for the comparison (e.g.identical amino acid, conservative amino acid substitution, etc.). Asthe term is used herein, two amino acids in question are considered asbeing “conservative substitutions” of one another if they each belong tothe same chemical class, i.e. acidic, nonpolar, uncharged polar andbasic. By way of non-limiting example, two different amino acidsbelonging to the class of nonpolar amino acids would be considered“conservative substitutions” of one another, even if these two aminoacids were not identical, whereas a nonpolar amino acid on the one handand a basic amino acid on the other hand would not be considered“conservative substitutions” of one another. Panel 3.1 of “MolecularBiology of the Cell”, 4^(th) Edition (2002), by Alberts, Johnson, Lewis,Raff, Roberts and Walter groups amino acids into four main groups:acidic, nonpolar, uncharged polar and basic. Such a grouping may be usedfor the purposes of determining, for the purposes of the presentinvention, whether or not a particular amino acid is a conservativesubstitution of another amino acid in question.

A further aspect of the invention provides a polynucleotide moleculehaving a nucleotide sequence encoding an amino acid sequence as set outin any of SEQ ID NOs: 1-48 and/or 52 to 56 or a nucleotide sequenceexhibiting at least 70% homology therewith, wherein homology may bedetermined by comparing a nucleotide sequence encoding an amino acidsequence of any of SEQ ID NOs: 1-48 and/or 52-56 with a nucleotidesequence in question by sequence alignment (as described above for aminoacid sequences), wherein a nucleotide in the sequence in question isconsidered homologous if it is either identical to the correspondingnucleotide in the nucleotide sequence encoding a corresponding aminoacid sequence of any of SEQ ID NOs: 1-48 and/or 52-56 or if one or morenucleotide deviation(s) in the sequence in question from thecorresponding one or more nucleotide(s) in the nucleotide sequenceencoding an amino acid sequence of any of SEQ ID NOs: 1-48 and/or 52-56results in a nucleotide triplet which, when translated, results in anamino acid which is either identical to (due to a degenerate triplet) ora conservative substitution of the corresponding amino acid in thecorresponding amino acid sequence of any of SEQ ID NOs: 1-48 and/or52-56. Here, the term “conservative substitution” is to be understood asdescribed above.

A further aspect of the invention provides a pharmaceutical compositioncomprising a human monoclonal antibody or fragment thereof or apolynucleotide molecule having a nucleotide sequence encoding an aminoacid sequence as set out in any of SEQ ID NOs: 1-48 and/or 52-56 orencoding an amino acid sequence comprising an amino acid sequencebearing at least 70% homology to any of SEQ ID NOs: 1-48 and/or 52-56,wherein “homology” is to be understood as explained hereinabove. Inaccordance with this invention, the term “pharmaceutical composition”relates to a composition for administration to a patient, preferably ahuman patient. In a preferred embodiment, the pharmaceutical compositioncomprises a composition for parenteral, transdermal, intraluminal,intraarterial, intrathecal and/or intranasal administration or by directinjection into tissue. It is in particular envisaged that saidpharmaceutical composition is administered to a patient via infusion orinjection. Administration of the suitable compositions may be effectedby different ways, e.g., by intravenous, intraperitoneal, subcutaneous,intramuscular, topical or intradermal administration. The pharmaceuticalcomposition of the present invention may further comprise apharmaceutically acceptable carrier. Examples of suitable pharmaceuticalcarriers are well known in the art and include phosphate buffered salinesolutions, water, emulsions, such as oil/water emulsions, various typesof wetting agents, sterile solutions, liposomes, etc . . . Compositionscomprising such carriers can be formulated by well known conventionalmethods. These pharmaceutical compositions can be administered to thesubject at a suitable dose. The dosage regimen will be determined by theattending physician and clinical factors. As is well known in themedical arts, dosages for any one patient depend upon many factors,including the patient's size, body surface area, age, the particularcompound to be administered, sex, time and route of administration,general health, and other drugs being administered concurrently.Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's, or fixedoils. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives may also be present such as,for example, antimicrobials, anti-oxidants, chelating agents, inertgases and the like. In addition, the pharmaceutical composition of thepresent invention might comprise proteinaceous carriers, like, e.g.,serum albumin or immunoglobulin, preferably of human origin. It isenvisaged that the pharmaceutical composition of the invention mightcomprise, in addition to the human monoclonal antibody or fragmentthereof (as described in this invention), further biologically activeagents, depending on the intended use of the pharmaceutical composition.Such agents might be drugs acting on the gastro-intestinal system, drugsacting as cytostatica, drugs preventing hyperurikemia, drugs inhibitingimmunoreactions (e.g. corticosteroids), drugs modulating theinflammatory response, drugs acting on the circulatory system and/oragents such as cytokines known in the art.

A further aspect of the invention provides a use of a human monoclonalantibody or fragment thereof as described hereinabove or apolynucleotide molecule comprising a nucleotide sequence encoding anamino acid sequence as set out in any of SEQ ID NOs: 1-48 and/or 52-56or encoding an amino acid sequence comprising an amino acid sequencebearing at least 70% homology to any of SEQ ID NOs: 1-48 and/or 52-56,wherein “homology” is to be understood as explained hereinabove, in themanufacture of a medicament, optionally comprising one or moreanti-inflammatory agents, for the treatment of inflammatory diseases.The inflammatory diseases are advantageously chosen from the groupconsisting of rheumatoid arthritis (RA) (including RA which is resistantto treatment with TNF-alpha neutralizers), asthma, multiple sclerosis(MS), chronic obstructive pulmonary disease (COPD), Acute RespiratoryDistress Syndrome (ARDS), Crohn's Disease, Idiopathic Pulmonary Fibrosis(IPF), Inflammatory Bowel Disease (IBD), uveitis, macular degeneration,colitis, psoriasis, Wallerian Degeneration, antiphospholipid syndrome(APS), acute coronary syndrome, restinosis, atherosclerosis, relapsingpolychondritis (RP), acute or chronic hepatitis, failed orthopedicimplants, glomerulonephritis, lupus or autoimmune disorders.

Of special interest is the use of the human monoclonal antibody orfragment thereof according to the invention for the preparation of amedicament for the treatment of RA (including RA which is resistant totreatment with TNF-alpha neutralizers), asthma, MS and/or Crohn'sdisease.

With regard to RA, asthma and/or MS, there are two popular theoriesregarding the pathogenesis of rheumatoid arthritis (RA). The first holdsthat the T cell, through interaction with an—as yetunidentified—antigen, is the primary cell responsible for initiating thedisease as well as for driving the chronic inflammatory process. Thistheory is based upon the known association of RA with class II majorhistocompatability antigens, the large number of CD4+ T cells and skewedT cell receptor gene usage in the RA synovium. GM-CSF is known toenhance antigen presenting function though increasing surface class IIMHC expression and GM-CSF is produced by T-cells, indicating a putativerole for GM-CSF in disease progression according to the T-cell basedhypothesis.

The second theory holds that, while T cells may be important ininitiating the disease, chronic inflammation is self-perpetuated bymacrophages and fibroblasts in a T-cell independent manner. This theoryis based upon the relative absence of activated T cells phenotypes inchronic RA and the preponderance of activated macrophage and fibroblastphenotypes. GM-CSF is a potent stimulator of macrophages and promotesproliferation of monocytes and macrophages.

GM-CSF known to be produced primarily by “effector” cells (macrophages)and connective tissue cells (fibroblasts) is expressed in abundance inRA synovium and synovial fluid, as measured by ELISA or mRNA studies.According to the “macrophage-fibroblast theory” of RA, these two celltypes appear to be largely responsible for creating a self-perpetuatingstate of chronic inflammation in which T cell participation may nolonger be critical. In this scenario, the activated macrophagecontinuously secretes IL-1 and TNF which maintain the synovialfibroblast in an activated state. The fibroblast, in turn, secreteslarge amounts of: a) cytokines—IL6, IL8 and GM-CSF; b) prostaglandins;and c) protease enzymes. GM-CSF feeds back to promote the maturation ofnewly recruited monocytes to macrophages. IL-8 and IL-6 contribute tothe recruitment and/or activation of yet other cell populations, whilethe prostaglandins and proteases act directly to erode and destroynearby connective tissues such as bone and cartilage.

With regard to Crohn's disease, recombinant human granulocyte/macrophagecolony stimulating factor (rGM-CSF) from yeast has shown efficacy in thetreatment of moderate to severe Crohn's disease (Dieckgraefe BK,Korzenik JR (2002). Lancet 360, 1478-80). Several review articles havesince then speculated about the therapeutic effect of this potentpro-inflammatory cytokine in this disease, believed to be of aninflammatory nature. Possible explanations for the mode of action ofrGM-CSF included an immunodeficiency component in Crohn's Disease, Th 2skewing, and expansion of dendritic cells promoting differentiation ofregulatory T cells (Wilk N J, Viney J L (2002). Curr Opin Invest Drugs3, 1291-6; Folwaczny C et al. (2003). Eur J Gastroenterol Hepatol 15,621-6). The inventors believe that a simpler mode of action, which atthe same time is more consistent with the known role of GM-CSF in otherpro-inflammatory diseases may be proposed.

GM-CSF is one of the most potent adjuvants known, which is why thecytokine is co-administered in numerous ongoing vaccination trials. Atthe same time, GM-CSF is highly immunogenic (Ragnhammar P et al. (1994).Blood 84, 4078-87). A very recent study (Rini B et al. (2005) Cytokine29, 56-66) has shown that daily subcutaneous treatment with rGM-CSF fromyeast, as performed in the Crohn's disease trial (Dieckgraefe B K,Korzenik J R (2002). Lancet 360, 1478-80), led within three months in87% (13/15) of prostate cancer patients, to the development ofantibodies against the cytokine. Sixty percent of patients (9/15)developed (polyclonal) GM-CSF neutralizing antibodies. The possibilityof a neutralizing response to GM-CSF was not investigated in the Crohn'sdisease trial, nor were serum levels of GM-CSF determined under therapy.Within the scope of this embodiment of the invention, it is contemplatedthat Crohn's disease patients treated with rGM-CSF did not directlyrespond only to the immune-stimulatory activity of the cytokine butalso, at least in part, responded clinically to an antibody responseneutralizing both the administered as well as the endogenous GM-CSF,which is known to be overproduced in Crohn's disease (Agnholt J et al.(2004) Eur J Gastroenterol Hepatol 16, 649-55). Neutralizing anti-GM-CSFantibodies, then, may have a similar therapeutic activity in Crohn'sdisease as does rGM-CSF, and should be considered as an alternativetherapeutic approach, as is contemplated hereinabove.

A further aspect of the invention provides a use of a human monoclonalantibody or fragment thereof as described hereinabove or apolynucleotide molecule comprising a nucleotide sequence encoding anamino acid sequence as set out in any of SEQ ID NOs: 1-48 and/or 52-56or encoding an amino acid sequence comprising an amino acid sequencebearing at least 70% homology to any of SEQ ID NOs: 1-48 and/or 52-56,wherein “homology” is to be understood as explained hereinabove in themanufacture of a medicament, optionally comprising one or moreadditional anti-cancer agents, for the treatment of a tumorous diseaseor another condition with delayed cell apoptosis, increased cellsurvival or proliferation. A preferred tumorous disease is a cancer, ofwhich leukaemia, multiple myeloma, gastric carcinoma or skin carcinomaare especially preferred.

Olver et al. ((2002) Cancer Chemother Pharmacol. 50, 171-8)subcutaneously applied the GM-CSF antagonist E21R in patients with solidtumors known to express GM-CSF receptors, leading to only a temporaryreduction of the PSA serum levels. Further, the application of thisGM-CSF antagonist in acute myeloid leukemia (“AML”) did not revealclinical activity (Jakupovic et al. (2004) Blood 103, 3230-2.). Stillfurther, the application of anti-GM-CSF monoclonal antibodies to AMLpatients did not reveal an anti-leukemic effect despite sufficient serumlevels and biological activity of the antibody in vivo (Bouabdallah etal. (1998) Leuk Lymphoma 30, 539-49). The authors thus concluded thattreatment with anti GM-CSF antibodies is not effective in AML patients.

The invention will now be described in more detail in the followingnon-limiting examples and figures, an overview of which follows:

FIG. 1 Absorption intensity (directly proportional to binding strength)for a variety of anti-rhGM-CSF scFv molecules obtained after four orfive rounds of panning in phage display as determined by ELISA

FIG. 2 Mean fluorescence intensity (inversely proportional toneutralization strength) for a variety of anti-rhGM-CSF scFv and othertest molecules as determined by a flow cytometry-based assay

FIG. 3 Results of a TF-1 proliferation inhibition assay performed usingthe anti-rhGM-CSF scFv molecule 5-306

FIG. 4 Absorption intensity (directly proportional to binding strength)for a variety of human anti-rhGM-CSF scFv molecules obtained after fouror five rounds of panning in phage display as determined by ELISA

FIG. 5 Results of a TF-1 proliferation inhibition assay performed usingvarious representative human anti-rhGM-CSF scFv hits

FIGS. 6A-C Binding specificity of human monoclonal antibodies for humanGM-CSF and other human colony stimulating factors

FIGS. 7A-I Surface plasmon resonance measurements characterizing kineticbinding of human monoclonal anti-GM-CSF antibodies and fragments thereof

FIG. 8A Sequence alignment of non-human primate GM-CSF and human GM-CSF

FIG. 8B Peptide spot radiogram showing binding of a fragment of a humanmonoclonal anti-GM-CSF to human GM-CSF

FIGS. 9A-B Qualitative results of a TF-1 proliferation inhibition assayperformed using various representative human anti-rhGM-CSF scFv antibodyfragments

FIGS. 10A-D Qualitative results of the TF-1 proliferation inhibitionassay performed using various representative human anti-rhGM-CSF IgGsand corresponding scFv fragments

FIG. 11 Quantitative results of the IL8 production assay performed usingvarious representative human anti-rhGM-CSF scFv antibody fragments

FIGS. 12A-B Quantitative results of the TF-1 proliferation inhibitionassay performed using various representative human anti-macGM-CSF IgGsand corresponding scFv fragments

FIG. 13 Results of comparative binding study showing selectivity ofbinding of anti-GM-CSF antibody IgG B for recombinant human GM-CSF andGM-CSF from various non-primate species

FIGS. 14A-B Results of assay investigating the dependence ofneutralizing potential of anti-GM-CSF antibody IgG B on theglycosylation of GM-CSF

FIGS. 15A-B Results of study of the effect of anti-GM-CSF antibody IgG Bon GM-CSF-mediated eosinophil survival

FIGS. 16A-B Results of study of the effect of anti-GM-CSF antibody IgG Bon GM-CSF-mediated eosinophil activation

FIGS. 17A-F Results of ex vivo toxicology study using anti-GM-CSFantibody IgG B, measured based on phagocytosis (FIGS. 17A-C) andoxidative burst (FIGS. 17D-F) by granulocytes

FIGS. 18A-F Results of ex vivo toxicology study using anti-GM-CSFantibody IgG B, measured based on phagocytosis (FIGS. 18A-C) andoxidative burst (FIGS. 18D-F) by monocytes

EXAMPLES Example 1 Procurement of the Recombinant Human (“rh”) GM-CSFAntigen used for the Generation of Neutralizing Human Antibodies andFragments Thereof Example 1.1 Cloning, Expression and Purification ofthe rhGM-CSF Antigen

The gene encoding human GM-CSF antigen was subcloned into the pET22b(+)vector (Novagene, USA) from the expression vector pORF-hGM-CSF (Novagen,USA) via the PCR-introduced restriction enzyme recognition sites NdeIand XhoI. The hGM-CSF-encoding gene in pET22b(+) was fused to the pelBleader sequence and is suitable for expression in E. coli periplasm.

Protein production and purification was performed as described by themanufacturer. In brief, E. coli BL21DE3 were transformed with theexpression plasmid and grown at 37° C. in selective medium to an opticaldensity of 0.5-0.8 at 600 nm. Protein production was induced by additionof IPTG to 1 mM and reduction of temperature to 25° C. A periplasmicpreparation was performed by osmotic shock using 20% sucrose solution toselectively destroy the cell wall maintaining an intact cell membrane.Native hGM-CSF contains two disulfide bridges and expression in theoxidative periplasm of E. coli allows for formation of thesefunctionally important disulfide bridges.

Recombinant human GM-CSF (“rhGM-CSF”) was purified in a two steppurification process via immobilized metal affinity chromatography(IMAC) and gel filtration. An Äkta® FPLC System (Pharmacia) and Unicorn®Software were used for chromatography. All chemicals were of researchgrade and purchased from Sigma (Deisenhofen) or Merck (Darmstadt).

IMAC was performed using a Qiagen Ni-NTA Superflow column according tothe protocol provided by the manufacturer. The column was equilibratedwith buffer A2 (20 mM sodium phosphate pH 7.2, 0.4 M NaCl) and theperiplasmic preparation (“PPP”) (100 mL) was applied to the column (2mL) at a flow rate of 2 mL/min The column was washed with 5 columnvolumes 5% buffer B2 (20 mM sodium phosphate pH 7.2, 0.4 M NaCl, 0.5 Mimidazole) to remove unbound sample. Bound protein was eluted using 100%buffer B2 in 5 column volumes. Eluted protein fractions were pooled forfurther purification.

Gel filtration chromatography was performed on a Superdex 200 Prep Gradecolumn (Pharmacia) equilibrated with PBS (Gibco). Eluted protein samples(flow rate 1 mL/min) were subjected to standard SDS-PAGE and WesternBlot for detection. Prior to purification, the column was calibrated formolecular weight determination (molecular weight marker kit, Sigma MWGF-200). Protein concentrations were determined measuring OD 280 nm andcalculated using the sequence-specific molecular extinction coefficient.

Example 1.2 Biotinylation of the rhGM-CSF Antigen

For phage library selection rhGM-CSF antigen produced in E. coli (seeabove) was biotinylated. Biotinylation was accomplished in PBScontaining 5% DMSO (Sigma) with a five-fold molar excess of EZ-LinkSulfo NHS-LC-LC-Biotin (Pierce) for 1 hour at room temperature in asample mixer (Dynal). For the separation of free Biotin and biotinylatedrhGM-CSF antigen, anion exchange chromatography (Resource Q, AmershamBiosciences) was carried out according to standard protocols. Thechromatography resulted in both approaches (designated A and B,described below) in two elution peaks. In case A the primary eluted peakwas fractionated again via a second anion exchange chromatography step(same conditions as above) into two elution peaks. Afterwards theobtained fractions were serially diluted (dilutions 1:2; startconcentration 6 μg/mL determined from the peak height) coated to 96wells ELISA plates and detected. The detection was carried out using A)an anti-human GM-CSF antibody M500-A (Sigma, 2.5 μg/mL in PBS/1% BSA)detected with horseradish peroxidase-conjugated goat anti-mouse Fab2specific polyclonal antibody (Dianova, 1 μg/mL PBS/1% BSA) and B) thematernal antibody (1 μg/mL PBS/1% BSA) detected with horseradishperoxidase-conjugated goat anti-rat polyclonal antibody (Dianova, 1μg/mL PBS/1% BSA). The successful biotinylation was demonstrated by asimilar ELISA experiment that was carried out using horseradishperoxide-conjugated streptavidin (Dako, 1 μg/mL PBS/1% BSA). The signalwas developed by adding OPD substrate solution (Sigma) and detected at awavelength of 492 nm (reference wavelength 620 nm). To estimate thedegree of biotinylation the above mentioned ELISA was carried out usingthe anion exchange fractions directly or after an incubation step with6.7×10 exp7 streptavidin magnetic beads (Dynabeads M-280-Streptavidin,Dynal) with gentle agitation for 30 minutes. The resulting supernatantwas coated onto the wells of 96-well ELISA plates and detected asdescribed above. The ELISA results showed that the second eluted peakcontained the biotinylated rhGM-CSF and that about 95% of the elutedrhGM-CSF was conjugated. Concentrations were estimated using theoriginal material as a standard and turned out to be about 20 μg/mL.

The retained bioactivity of the biotin-labeled rhGM-CSF was confirmed inTF-1 proliferation assays according to protocols described below in thecharacterization of the single chain antibodies (scFvs).

Example 1.3 Fluorescein Labeling of the rhGM-CSF antigen

For binding studies on TF-1 cells recombinant human GM-CSF antigenproduced in E. coli (see Example 1.2 above) was conjugated withfluorescein-5(6)-carboxamidocaproic acid N-succinimidyl ester (Fluka,fluorescein-NHS). The conjugation step was performed in borate buffer(0.05 M boric acid, 0.1 M NaCl, pH 8.5) containing 17.5% DMSO with afive-fold molar excess of fluorescein-NHS for 1 hour at room temperaturein a sample mixer. Afterwards, gel filtration (Sephadex G25, AmershamBiosciences) was carried out to dissociate fluorescein-labeled rhGM-CSFantigen from free fluorescein-NHS. The gel filtration resulted in twopeaks measured at a wavelength of 485 nm (reference wavelength 535 nm),whereas the primary peak represents the FITC-labeled rhGM-CSF. Thedegree of labeling was determined by defining the F/P ratio of theconjugate ([mg/mL]=(A₂₈₀−0.35×A₄₉₃)×1.08;F/P=(A₄₉₃/73.000)×(15.000/([mg/mL])). The determined concentration was0.041 mg/mL with an F/P ratio of 1.2.

Example 2 Generation and Selection of Neutralizing Human Anti-GM-CSFAntibodies and Fragments Thereof Example 2.1 Cloning of the Maternal VHfrom Hybridoma HB-9569

As used throughout the foregoing examples, a “maternal” V-region denotesthat the V-region in question originates from a full immunoglobulinmolecule.

As used throughout the foregoing examples, a “hit” denotes a moleculewhich is known to bind an antigen of interest, but which binding has notbeen quantitatively evaluated. A “hit” is a molecule in an early stageof characterization for which small-scale production might have alreadybeen performed. Such a molecule is in the validation stage ofcharacterization.

As used throughout the foregoing examples, a “lead” molecule denotes amolecule the binding and neutralization potentials of which have beenquantified. Production of a “lead” molecule has already taken place on alarge scale.

In the following examples one possible way of generating a fully humanmonoclonal antibody neutralizer of human GM-CSF and generation offragments thereof is described.

The aim of this experiment is the isolation and sub-cloning of the geneencoding the VH in the maternal mAb produced by the hybridoma cell lineHB-9569. The hybridoma HB-9569 was obtained from ATCC (USA). Hybridomacells were cultivated in ATCC complete growth medium: RPMI 1640 mediumwith 2 mM L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate,4.5 g/L glucose, 10 mM HEPES, and 1.0 mM sodium pyruvate andsupplemented with 0.05 mM 2-mercaptoethanol, fetal bovine serum 10% at37° C. with 5% CO₂. For total RNA preparation, 1×10 exp7 cells were usedand RNA was prepared as described in the product manual of the QiagenOmni-Skript Kit (Qiagen, Germany). cDNA was synthesized according tostandard methods (Sambrook, Cold Spring Harbor Laboratory Press 1989,Second Edition).

For the isolation of heavy chain V-region DNA, RT-PCR was carried outusing MHALT1R.V: GCC GAA TTC CAC CAT GGR ATG SAG CTG KGT MAT SCT CTT andRace GSP rIgG2a/b: CAC ACC GCT GGA CAG GGC TCC AGA GTT CC primer set.The following PCR program was used for amplification: Denaturation at94° C. for 15 seconds, primer annealing at 52° C. for 50 seconds andprimer extension at 72° C. for 90 seconds were performed over 40 cycles,followed by final extension at 72° C. for 10 minutes. Heavy chain DNAV-fragments were then isolated according to standard protocols.

The heavy chain DNA V-fragment was cloned into PCR script-CAM(Stratagene) as described by the manufacturer. The sequences wereidentified by sequencing.

Example 2.2 Selection of a Human VL

The aim of this experiment is the selection of a human VL which can pairwith the maternal VH cloned as described above.

Example 2.2.1 Isolation of RNA from Selected IgD-Positive B-Cells

100 mL blood were taken from five healthy human donors. Peripheral bloodmononuclear cells (PBMCs) were isolated by a ficoll-gradient accordingto standard methods. To select IgD-positive cells, 1 mL anti-mouseIgG-beads (CELLection™ Pan Mouse IgG Kit; DYNAL) were coated with 20 μgmouse anti-human IgD-antibody (PharMingen). Approximately 2.5×10 exp7PBMCs were added to the beads and incubated at 4° C. for 15 minutes.After washing four times with 1 mL RPMI-medium (BioChrom) IgD-positivecells were released from the beads by adding 8 μL release buffer (DNase)and transferred to a fresh tube. By this method 0.9×10 exp5 to 3.7×10exp6 IgD-positive cells could be obtained. Total RNA was isolated fromIgD-positive cells using the RNeasy® Midi Kit (QIAGEN) following themanufacturer's instructions. cDNA was synthesized according to standardmethods (Sambrook, Cold Spring Harbor Laboratory Press 1989, SecondEdition).

Example 2.2.2 PCR-Amplification of Variable Light Chain Regions(VL-Regions)

For the isolation of light chain V-region DNA, RT-PCR was carried outusing V-kappa-(5′-huVK1-SacI-2001 (5′-GAGCCGCACG AGCCCGAGCT CCAGATGACCCAGTCTCC-3′), 5′-huVK2/4-SacI-2001 (5′-GAGCCGCACG AGCCCGAGCT CGTGATGACYCAGTCTCC-3′), 5′-huVK3-SacI-2001 (5′-GAGCCGCACG AGCCCGAGCT CGTGWTGACRCAGTCTCC-3′), 5′-huVK5-SacI-2001 (5′-GAGCCGCACG AGCCCGAGCT CACACTCACGCAGTCTCC-3′), 5′-huVK6-SacI-2001 (5′-GAGCCGCACG AGCCCGAGCT CGTGCTGACTCAGTCTCC-3′), 3′-hu-Vk-J1 -SpeI-BsiWI (5′-GACGACACTA GTTGCAGCCACCGTACGTTT GATTTCCACC TTGGTCC-3′), 3′-hu-Vk-J2/4-SpeI-BsiWI(5′-GACGACACTA GTTGCAGCCA CCGTACGTTT GATCTCCASC TTGGTCC-3′),3′-hu-Vk-J3-SpeI-BsiWI (5′-GACGACACTA GTTGCAGCCA CCGTACGTTT GATATCCACTTTGGTCC-3′), 3′-hu-Vk-J5-SpeI-BsiWI (5′-GACGACACTA GTTGCAGCCA CCGTACGTTTAATCTCCAGT CGTGTCC-3′) primer sets. RNA from IgD-positive B-cells wastranscribed into cDNA (as described above) and used as template DNA inPCR reactions. Per PCR reaction, one 5′-primer was combined with one3′-primer. The number of different PCR reactions was determined by thenumber of possible combinations of 5′- and 3′-primers. The followingPCR-program was used for amplification: Denaturation at 94° C. for 15seconds, primer annealing at 52° C. for 50 seconds and primer extensionat 72° C. for 90 seconds were performed over 40 cycles, followed byfinal extension at 72° C. for 10 minutes. Light chain DNA V-fragmentswere then isolated according to standard protocols.

Example 2.2.3 Library Construction—Cloning of the Human VL Pool

A phage display library was generally constructed based on standardprocedures, as for example disclosed in “Phage Display: A LaboratoryManual”; Ed. Barbas, Burton, Scott & Silverman; Cold Spring HarborLaboratory Press, 2001.

The primers chosen for PCR amplification gave rise to 5′-SacI and3′-SpeI recognition sites for the light chain V-fragments. Two ligationreactions were set up, each consisting of 400 ng of the kappa lightchain fragments (SacI-SpeI digested) and 1400 ng of the plasmidpBluescript KS+(SacI-SpeI digested; large fragment). The two resultingantibody V-light chain pools were then each transformed into 300 μL ofelectrocompetent Escherichia coli XL1 Blue by electroporation (2.5 kV,0.2 cm gap cuvette, 25 mF, 200 Ohm, Biorad gene-pulser) resulting in alibrary size of 5.8×10 exp8 independent clones.

Kappa (light chain) DNA-fragments from the different PCR amplificationswere weighted for each ligation as follows: Each 5′-primer defines aspecific group. Within these groups the 3′-primers define the subgroups.The subgroups were weighted 1:2:1:1 corresponding to the primers3′-hu-Vk-J1-SpeI-BsiWI:3′-hu-Vk-J2/4-SpeI-BsiWI:3′-hu-Vk-J3-SpeI-BsiWI:3′-hu-Vk-J5-SpeI-BsiWI.The groups were weighted according to their germline distribution1:1:1:0.2:0.2 corresponding to the primers5′-huVKl-Sac-2001:5′-huVK3-Sac-2001:5′-huVK2/4-Sac-2001:5′-huVK5-Sac-2001:5′-huVK6-Sac-2001.

After electroporation the assay was incubated in SOC broth (Fluka) forphenotype expression. The cultures were then each incubated in 500 mL ofSB selection medium containing 50 μg/mL carbenicillin and 2% w/v glucoseovernight. The next day, cells were harvested by centrifugation andplasmid preparation carried out using a commercially available plasmidpreparation kit (Qiagen).

Example 2.2.4 Construction of the Antibody Library—Human VL—Maternal VH

PCR was performed to amplify the maternal VH from the vector containingthe maternal VH described above in Example 2.1. For amplification a PCRprotocol according to standard procedures was followed using the5′-primer MVH8 (5′-GAG GTT CAG CTC GAG CAG TCT GGA GCT-3′) and the3′-primer 3′-MuVHBstEII (5′-TGA GGA GAC GGT GAC CGT GGT CCC TTG GCC CCAG-3′).

After purification of the approximately 350 by amplification productfrom an analytical agarose gel, the DNA fragment was cut with therestriction enzymes BstEII and XhoI. The phagemid pComb3H5BHis (thisvector is described in the thesis dissertation of Dr. Ralf Lutterbüse)was digested accordingly and the large fragment was ligated with theabove mentioned fragment. After transformation into E. coli XL1 blue, asingle clone was cultivated in 100 mL SB medium (containing 50 μg/mLcarbenicilline) and the plasmid was prepared according to standardprotocols. The successful cloning was confirmed by sequencing the insert(Sequiserve, Munich).

This vector pComb3H5BHis/maternalVH was restricted with the restrictionenzymes SacI and SpeI. The large vector fragment was isolated.Plasmid-DNA containing the VK-library from Example 2.2.3 was restrictedwith the restriction enzymes SacI and SpeI. The small VK fragment band(approx 350 bp) was isolated.

1200 ng of the vector fragment were ligated with 400 ng of the VKfragments and transformed into 300 μL of electrocompetent E. coli XL1Blue by electroporation (2.5 kV, 0.2 cm gap cuvette, 25 mF, 200 Ohm)resulting in a total scFv library size of 2.8×10 exp8 independentclones.

After phenotype expression and slow adaptation to carbenicillin, theantibody library was transferred into SB-Carbenicillin (50 μg/mL)selection medium. The antibody library was then infected with aninfectious dose of 1×10 exp12 particles of helper phage VCSM13 resultingin the production and secretion of filamentous M13 phage, wherein eachphage particle contained single stranded pComb3H5BHis-DNA encoding ahalf-human scFv-fragment and displayed the corresponding scFv-protein asa translational fusion to phage coat protein III.

Example 2.2.5 Phage Display Selection of a Human VL

The phage particles carrying the scFv-repertoire were harvested from theculture supernatant by PEG8000/NaCl precipitation and centrifugation.Then approximately 1×10 exp 11 to 1'10 exp12 scFv phage particles wereresuspended in 0.4 mL of PBS/0.1% BSA and incubated with recombinantbiotinylated soluble rhGM-CSF (produced in E. coli as described above inexample 1) for 2 h with gentle agitation in a total volume of 0.5 mL(Antigen concentrations Rounds 1-3: 100 nM; round 4: 10 nM; round 5: 1nM). Then 6.7×10 exp7 streptavidin magnetic beads (DynabeadsM-280-Streptavidin, Dynal) were added and further incubated under gentleagitation for 30 minutes.

scFv phage that did not specifically bind to the target antigen wereeliminated by washing steps with PBS/0.1% BSA. For that purpose thebiotinylated antigen—streptavidin bead complexes (with the potentialscFv binders) were collected with a magnet and resuspended in 1 mL ofthe washing solution (one washing step). This washing procedure wasrepeated up to four times in further rounds.

After washing, binding entities were eluted by using HCl-glycine, pH2.2. Following neutralization with 2 M Tris, pH 12, the eluate was usedfor infection of a fresh uninfected E. coli XL1 Blue culture. To eluteremaining high binding entities this step was repeated usingHCl-glycine, pH 1.0. This second eluate was again neutralized and usedfor infection of a fresh uninfected E. coli XL1 Blue culture. Bothinfected E. coli cultures were then mixed and cells that weresuccessfully transduced with a phagemid copy, encoding a humanscFv-fragment, were again selected for carbenicillin resistance andsubsequently infected with VCSM13 helper phage to start the second roundof antibody display and in vitro selection.

Plasmid DNA corresponding to 4 and 5 rounds of panning was isolated fromE. coli cultures. For the production of soluble scFv-protein, VL-DNAfragments were excised from the plasmids (SacI-SpeI), and cloned via thesame restriction sites in the plasmid pComb3H5BFlag/His with thematernal VH differing from the initial pComb3H5BHis/maternal VH in thatthe expression construct (e.g. scFv) includes a Flag-tag (TGDYKDDDDK)between the scFv and the His6-tag and the additional phage proteins aredeleted.

After ligation each pool (different rounds of panning) of plasmid DNAwas transformed into 100 μL heat shock competent E. coli XL1 blue andplated onto carbenicillin LB-agar. Single colonies were picked into 100μL of LB carb (50 μg/mL).

10 μl of this cell suspension was typically incubated in 5 ml SB mediumsupplemented with carbenicillin to a concentration of 50 μg/ml and MgCl₂to a final concentration of 20 mM for approximately 6 h at 37° C. underagitation. Then IPTG was added to a final concentration of 1 mM and theincubation continued overnight on a shaker at 30° C.

Cells were centrifuged to a pellet and this pellet was typicallyresuspended in 0.5 ml PBS. By four rounds of freezing at −70° C. andthawing at 37° C., the outer membrane of the bacteria was destroyed byosmotic shock and the soluble periplasmic proteins including the scFvswere released into the supernatant. After elimination of intact cellsand cell-debris by further centrifugation (5 min at 10,000×g), thesupernatant (i.e. PPP) containing the scFvs was collected and examinedfurther.

RhGM-CSF antigen (Leukine Liquid, Immunex) was immobilized on ELISAplates overnight at 4° C. (50 μl of 1 μg antigen/ml PBS per well). Afterwashing the wells one time with PBS and blocking with PBS 3% BSA for 1 hat room temperature 100 μl PPPs containing scFvs were added to the wellsand typically incubated for 1 h at room temperature. After three washeswith PBS/0.05% Tween20, detection of scFv-fragments bound to immobilizedantigen was carried out using an anti-flag M2 (1 μg/mL PBS/1% BSA) anddetected with horseradish peroxidase-conjugated goat anti mouse Fab2specific polyclonal antibody (Dianova, 1 μg/mL PBS/1% BSA). The signalwas developed by adding 2,2′-azino-di[3-ethyl-benzthiazoline-6-sulphonic acid] (“ABTS”) substrate solutionand detected at a wavelength of 405 nm according to standard protocols.

From 20 clones tested (10 obtained after 4 rounds and 10 obtained after5 rounds of panning), five lysates showed strong ELISA signals incontrast to PBS as a negative control on the recombinant antigen. ELISAresults are shown in FIG. 1, in which the various scFv molecules testedare arrayed along the x-axis and the y-axis shows the absorptionintensity measured, with higher absorption indicating stronger binding.The PBS negative control is indicated on the x-axis at the far left.ScFv molecules exhibiting appreciable binding are denoted with above therespective absorption intensity column either a diamond or an asterisk.The diamond and asterisk in FIG. 1 represent two different sequences,i.e. the scFv whose absorption intensity column is indicated with adiamond was of one sequence, whereas all scFvs whose absorptionintensity columns are indicated by asterisks share the same commonsequence.

The five ELISA-positive clones were subjected to DNA sequencing.Sequencing was carried out at Sequiserve (Munich). A total of fourclones shared the DNA sequence corresponding to scFv 5-306 while theother sequence (4-301) was identified only once. The dominant sequencecorresponding to scFv 5-306 as well as the sequence 4-301 were of humanorigin and displayed very close homology to human germ line sequenceVk1-O12.

Example 2.2.6 Characterization of scFv Hit Constructs Derived from thehuVL Selection

The aim of the following experiments was the characterization of thescFv hits generated by the methods described above.

Example 2.2.6.1 Small-scale expression and purification of scFv hits(derived as described above) in E. coli

To obtain PPPs the cells were grown in SB-medium supplemented with 20 mMMgCl₂ and carbenicillin 50 μg/mL and were redissolved in 1 mL PBS afterharvesting. The outer membrane of the bacteria was destroyed bytemperature shock (four rounds of freezing at −70° C. and thawing at 37°C.) and the soluble periplasmic proteins including the scFvs werereleased into the supernatant. After elimination of intact cells andcell-debris by centrifugation, the supernatants containing the scFvswere collected and examined further. For further purification, 25 μL 20mM NaH₂PO₄, 400 mM NaCl, 250 mM imidazole, pH 7.0 was added to arespective PPP. The PPP was purified via Ni-NTA Spin Columns (Qiagen) asrecommended in the manual. In brief, a respective PPP solution was addedto the pre-equilibrated column to bind to the resin. The Spin Columnswere washed twice with 20 mM NaH₂PO₄, 400 mM NaCl, 20 mM imidazole, pH7.0. The bound protein was eluted twice in 200 μL 20 mM NaH₂PO₄, 400 mMNaCl, 250 mM imidazole, pH 7.0. The purified scFv proteins were furtheranalyzed with respect to binding strength (kinetic off rate) andneutralization capabilities (inhibition of GM-CSF dependent TF-1proliferation) as described in the subsequent examples. Though notseparating and distinguishing between the different possibleconformations of the scFv, this crude purification of PPP yields 80%pure scFv protein as judged by Western-blot analysis (data not shown).

Example 2.2.6.2 Inhibition of FITC-Labelled rhGM-CSF

The aim of this experiment is to show that the identified scFv clonesare capable of inhibiting binding of rhGM-CSF to the GM-CSF receptorcomplex displayed on the surface of TF-1 cells. Neutralizing scFvconstructs would be expected to compete for the receptor-binding epitopeon the rhGM-CSF molecule, rendering it impossible for rhGM-CSF to bindto the GM-CSF receptor complex. To the extent that binding by rhGM-CSFto its receptor is inhibited in the above manner, one would expect toobserve a decrease in the intensity of fluorescence staining of TF-1cells by fluorescein-labelled rhGM-CSF (rhGM-CSF-FITC) in a flowcytometry-based assay.

The following describes the performance of such a flow cytometry-basedassay. A final concentration of 0.4 μg/mL rhGM-CSF-FITC conjugate in PBSwas incubated with 10 μg/ml of the maternal antibody or undilutedperiplasmic extract of the scFv that had been purified with NiNTA SpinColumn. The protein samples were left to equilibrate at 25° C. for 1 hprior to addition of a suspension of TF-1 cells. The TF-1 cells werecultivated in RPMI 1640 medium (Gibco; L-glutamine, phenol-red free),10% heat-inactivated FCS in the absence of rhGM-CSF overnight. A finalconcentration of 2×10 exp6 cells/mL and 150 μL of cell suspension wereused per sample. The cells were harvested by centrifugation at 500×g at4° C. for 3 min and washed twice with FACS buffer. The washed cells wereresuspended in 100 μL of pre-equilibrated protein sample containing therhGM-CSF-FITC and respective maternal antibody or scFv. The samples wereincubated at 4° C. for 60 min. After two further washes the cells wereresuspended in 150 μL ice cold FACS buffer and subsequently analysed byflow cytometry. The results are shown in FIG. 2. Specifically, FIG. 2shows a graph in which various test molecules are arrayed along thex-axis, and in which the mean fluorescence intensity (“MFI”) isindicated on the y-axis. As can be seen in FIG. 2, a clear loss offluorescence intensity of the TF-1 cells was observed with the maternalantibody (second from left along the x-axis). The competition binding torhGM-CSF of the scFv molecule designated 5-306 could be monitored byloss in fluorescence staining of TF-1 cells while the scFv molecule4-301 hardly showed any effect. Since these results suggest that thescFv molecule designated 5-306 may be a promising neutralizer of GM-CSF,further analysis of the neutralizing activity was restricted to scFv5-306.

Example 2.2.6.3 Large Scale Expression and Purification of scFv Lead

Protein production and purification on a large scale was performed asfollows. In brief, E. coli BL21DE3 were transformed with the expressionplasmid and grown at 37° C. in 1 L selective medium to an opticaldensity at 600 nm of 0.5-0.8. Protein production was induced by additionof IPTG to 1 mM and the cultures were incubated for another 16 h withagitation at a temperature of 25° C. The cells were harvested bycentrifugation at 5,000×g for 10 mM and resuspended in 100 mL 1×PBS.Periplasmic proteins were extracted by optimal, sequential freezing inethanol/dry ice and thawing in a 37° C. water bath over four cycles.Finally, the extract was centrifuged at 10,000×g for 20 min.

The scFv 5-306 was isolated in a two-step purification process ofimmobilized metal affinity chromatography (IMAC) and gel filtration. Allleads were purified according to this method. Äkta® FPLC System(Pharmacia) and Unicorn® Software were used for chromatography. Allchemicals were of research grade and purchased from Sigma (Deisenhofen)or Merck (Darmstadt).

IMAC was performed using a Qiagen Ni-NTA Superflow column according tothe protocol provided by the manufacturer. The column was equilibratedwith buffer A2 (20 mM sodium phosphate pH 7.2, 0.4 M NaCl) and the PPP(100 mL) was applied to the column (2 mL) at a flow rate of 2 mL/min Thecolumn was washed with 5 column volumes 5% buffer B2 (20 mM sodiumphosphate pH 7.2, 0.4 M NaCl, 0.5 M imidazole) to remove unbound sample.Bound protein was eluted using 100% buffer B2 in 5 column volumes.Eluted protein fractions were pooled for further purification.

Gel filtration chromatography was performed on a HiLoad™ 16/60 Superdex75 Prep Grade column (Pharmacia) equilibrated with PBS (Gibco). Elutedprotein samples (flow rate 1 mL/min) were subjected to standard SDS-PAGEand Western Blot for detection. Prior to purification, the column wascalibrated for molecular weight determination (molecular weight markerkit, Sigma MW GF-200). The size dependent separation on the Superdex 75Prep Grade column resulted in clearly distinguishable monomer andassociative dimer peak fractions of the scFv leads. Proteinconcentrations were determined measuring optical density at 280 nm andwere calculated using the sequence-specific molecular extinctioncoefficient of the respective scFv lead.

Example 2.2.6.4 Inhibition of rhGM-CSF-Dependent Proliferation of TF-1Cells by an scFv Lead

The aim of this experiment is to achieve qualitative information on theneutralizing activity of the half-human scFv 5-306 using the hGM-CSFdependant cell line TF-1 (DSMZ ACC 334). TF-1 cells were cultivated inRPMI 1640 medium (Gibco; L-glutamine, phenol-red free), 10% heatinactivated FCS in the presence of 2.5 ng/mL rhGM-CSF as described bythe distributor (Deutsche Sammlung von Mikroorganismen and ZellkulturenGmbH, Braunschweig, Germany). Cells were grown to a cell density of0.5×10 exp6 cells/mL. For the proliferation assay TF-1 cells wereharvested by centrifugation at 300×g for 4 min and washed with 1×PBS(Dulbecco's, Gibco). Cells were resuspended at a final concentration of1×10 exp5 cells/mL in RPMI 1640, 10% FCS and 90 μL cell suspension perMicrotest flat bottom cell culture plate well were used (0.9×10 exp4cells/well). A final concentration of 0.3 ng/mL rhGM-CSF was used tostimulate the proliferation of the TF-1 cells. For neutralization ofhGM-CSF dependent proliferation 10 μL of purified scFv were added to 100μL TF-1 and rhGM-CSF solution in a dilution series ranging fromapproximately 100 μg/ml to 100 pg/ml. The samples were incubated at 37°C. at 5% CO₂ for 72 h. After 72 h the proliferative status of the TF-1cells was determined adding WST-1 and monitoring the colorimetric changewith an ELISA reader at 450 nm. The data were fitted for half maximalinhibition of proliferation (IC₅₀) using the non-linear regression curvefit of the Prism software.

A clearly dose-dependant proliferation inhibiting effect of scFv 5-306could be seen and was comparable for the monomeric and the dimericconformational forms. By fitting for the half-maximal inhibition ofproliferation an IC50 value of 7.3 nM was determined for the monomericform and 3.5 nM for the dimeric form. The results are shown in FIG. 3.

Example 2.3 Construction of the Antibody Libraries and Phage DisplaySelection of Human VHs

The aim of the following experiments is the selection of a set of humanVH regions that would pair with the human VL region of scFv 5-306selected as described above.

Example 2.3.1 Isolation of RNA from Peripheric Blood Mononuclear Cells(PBMCs)

100 mL blood were taken from five healthy human donors. Peripheral bloodmononuclear cells (PBMCs) were isolated by a ficoll-gradient accordingto standard methods. Total RNA was isolated from PBMCs using the RNeasy®Midi Kit (QIAGEN) following the manufacturer's instructions. cDNA wassynthesized according to standard methods (Sambrook, Cold Spring HarborLaboratory Press 1989, Second Edition).

Example 2.3.2 PCR-Amplification of Variable Heavy Chain Regions(VH-Regions)

The VH library was constructed and named Lib 134-VH. This VH-libraryconsists of the human repertoire of FR1-CDR2-FR2-CDR2-FR3 from the PCRamplified VH-regions of the above described PBMC pool, linkedoperatively to the VH CDR3 of the maternal antibody followed by a humanFR4 germline sequence.

For the isolation of human template VH-regions, RT-PCR was carried outusing a 5′-VH-specific primer set (5′-huVH1,3,5-XhoI-2001 (5′-AGG TGCAGC TGC TCG AGT CTG G-3′), 5′-huVH4-XhoI-2001 (5′-CAG GTG CAG CTG CTCGAG TCG GG-3′), 5′-huVH4B-XhoI-2001 (5′-CAG GTG CAG CTA CTC GAG TGGGG-3′)) and a set of two 3′-VH-specific primers (3′-hu-VH-BstEII-2001(5′-CTG AGG AGA CGG TGA CC-3′), 3′-hu-VH-J3-BstEII-2001 (5′-CTG AAG AGACGG TGA CC-3′)). Per PCR reaction, one 5′-primer was combined with one3′-primer; the number of different PCR reactions was determined by thenumber of possible combinations of 5′- and 3′-primers. The PBMC cDNA (asdescribed above of four donors only was used as a source of VH-genes).The following PCR-program was used for amplification: Denaturation at94° C. for 15 seconds, primer annealing at 52° C. for 50 seconds andprimer extension at 72° C. for 60 seconds was performed over 40 cycles,followed by final extension at 72° C. for 10 minutes. The amplificationproducts with a size of approximately 350 by were isolated according tostandard methods.

For the isolation of Lib 134-VH-regions, RT-PCR was carried out in twosteps. First, the human heavy chain VH-segments (FR1-CDR1-FR2-CDR2-FR3)were PCR-amplified from the isolated template VH fragments using thesame 5′-VH-specific primer set as described above(5′-huVH1,3,5-XhoI-2001, 5′-huVH4-XhoI-2001, 5′-huVH4B-XhoI-2001) and a3′-specific primer set (3′-Lib 134-VH-1A-MH3 (5′-GTA ATC AAA GTA GAC TGCTAT CAG ACC CGA TCT YGC ACA GTA ATA CAC GGC-3′), 3′-Lib 134-VH-1B-MH3(5′-GTA ATC AAA GTA GAC TGC TAT CAG ACC CGA TCT YGC ACA GTA ATA CAYRGC-3′), 3′-Lib 134-VH-3A-MH3 (5′-GTA ATC AAA GTA GAC TGC TAT CAG ACCCGA TCT NGY ACA GTA ATA CAC RGC-3′), 3′-Lib 134-VH-3B-MH3 (5′-GTA ATCAAA GTA GAC TGC TAT CAG ACC CGA TCT NGC ACA GTA ATA CAA RGC-3′), 3′-Lib134-VH-4-MH3 (5′-GTA ATC AAA GTA GAC TGC TAT CAG ACC CGA TCT SGC ACA GTAATA CAC RGC-3′)) for the human VH subfamilies 1, 3 and 4 matching in thevery terminal region of FR3.

The following primer combinations were used:

-   a) 5′-huVH1,3,5-XhoI-2001×3′-Lib 134-VH-1A-MH3-   b) 5′-huVH1,3,5-XhoI-2001×3′-Lib 134-VH-1B-MH3-   c) 5′-huVH1,3,5-XhoI-2001×3′-Lib 134-VH-3A-MH3-   d) 5′-huVH1,3,5-XhoI-2001×3′-Lib 134-VH-3B-MH3-   e) 5′-huVH4-XhoI-2001×3′-Lib 134-VH-4-MH3-   f) 5′-huVH4B-XhoI-2001×3′-Lib 134-VH-4-MH3

Per PCR reaction, one 5′-primer was combined with the 3′-primer; thenumber of different PCR reactions was determined by the number ofpossible combinations of 5′- and the 3′-primer. The followingPCR-program was used for amplification: Denaturation at 94° C. for 15seconds, primer annealing at 52° C. for 50 seconds and primer extensionat 72° C. for 90 seconds was performed over 40 cycles, followed by finalextension at 72° C. for 10 minutes. Through this PCR step and therespective 3′-primer sequence, the human VH segments are prolonged for apart of the maternal VH CDR3, which then in turn is the priming site forthe second step PCR 3′-primer.

These VH-(FR1-CDR1-FR2-CDR2-FR3) DNA-fragments were then used astemplates in a second PCR reaction using again the respective5′VH-specific primer and a universal 3′ primer matching to the universal3′-terminus of the amplified DNA-fragments (3′-Lib 134-JH3-BstE2, 5′-AGAGAC GGT GAC CAT TGT CCC TTG GCC CCA GTA ATC AAA GTA GAC TGC-3′).

The following PCR-program was used for amplification:

Denaturation at 94° C. for 15 seconds, primer annealing at 52° C. for 50seconds and primer extension at 72° C. for 60 seconds were performedover 40 cycles, followed by final extension at 72° C. for 10 minutes.The DNA V-fragments were isolated according to standard protocols.

Example 2.3.3 Library Construction—Cloning of the Human VH Pool

In a second round of the foregoing method, the human VL of scFv 5-306identified in the first, previous selection was chosen, and subsequentlycombined with the library of human VH fragments described in Example2.3.2 with the aim of generating a human scFv. A phage display librarywas generally constructed based on standard procedures, as for exampledisclosed in “Phage Display: A Laboratory Manual”; Ed. Barbas, Burton,Scott & Silverman; Cold Spring Harbor laboratory Press, 2001.

Heavy chain DNA-fragments from the different PCR amplifications wereweighted for each ligation as follows:

a:b:c:d:e:f=3:1:3:1:1:1, wherein a-f have the following meanings:

-   a) 5′-huVH1,3,5-XhoI-2001×3′-Lib 134-VH-1A-MH3×3′-Lib 134-JH3-BstE2-   b) 5′-huVH1,3,5-XhoI-2001×3′-Lib 134-VH-1B-MH3×3′-Lib 134-JH3-BstE2-   c) 5′-huVH1,3,5-XhoI-2001×3′-Lib 134-VH-3A-MH3×3′-Lib 134-JH3-BstE2-   d) 5′-huVH1,3,5-XhoI-2001×3′-Lib 134-VH-3B-MH3×3′-Lib 134-JH3-BstE2-   e) 5′-huVH4-XhoI-2001×3′-Lib 134-VH-4-MH3×3′-Lib 134-JH3-BstE2-   f) 5′-huVH4B-XhoI-2001×3′-Lib 134-VH-4-MH3×3′-Lib 134-JH3-BstE2

One ligation reaction was set up consisting of 400 ng of human Lib134-VH fragment pool (XhoI-BstEII digested) and 1200 ng of the plasmidpComb3H5BHis/5-306 VL (the DNA encoding the VL region of scFv 5-306 wascloned via the restriction sites Sad and Spel into pComb3H5BHisaccording to standard procedures). The resulting antibody human VH poolwas then transformed into 300 μL of electrocompetent Escherichia coliXL1 Blue by electroporation (2.5 kV, 0.2 cm gap cuvette, 25 mF, 200 Ohm,Biorad gene-pulser) resulting in a library size of 1.6×10 exp8independent clones in total.

After electroporation the assay was incubated in SOC broth (Fluka) forphenotype expression. The cultures were then each incubated in 500 mL ofSB selection medium containing 50 μg/mL carbenicillin and 2% v/v glucoseovernight. The next day, cells of the cultures were harvested bycentrifugation and plasmid preparation carried out using a commerciallyavailable plasmid preparation kit (Qiagen) to preserve the DNA library.

1.5 μg of this plasmid pool encoding the respective scFv pool were thenelectroporated into E. coli XL1blue (2.5 kV, 0.2 cm gap cuvette, 25 mF,200 Ohm, Biorad gene-pulser) resulting in a library size of 2.4×10 exp9independent clones in total. After phenotype expression and slowadaption to carbenicillin the antibody library was transferred intoSB-Carbenicillin (50 μg/mL) selection medium. The antibody library wasthen infected with an infectious dose of 1×10 exp12 particles of helperphage VCSM13 resulting in the production and secretion of filamentousM13 phage, wherein each phage particle contained single strandedpComb3H5BHis-DNA encoding a human scFv-fragment and displayed thecorresponding scFv-protein as a translational fusion to phage coatprotein III.

Example 2.3.4 Phage Display Selection of a Human VH

The resulting phage library carrying the cloned scFv-repertoire washarvested from the culture supernatant by PEG8000/NaCl precipitation andcentrifugation. Approximately 1×10 exp11 to 1×10 exp12 scFv phageparticles were resuspended in 0.4 mL of PBS/0.1% BSA and incubated withrecombinant biotinylated soluble rhGM-CSF (E. coli material, asdescribed in example 1) for 1 h under gentle agitation in a total volumeof 0.5 mL. Then 6.7×10 exp7 streptavidin magnetic beads (DynabeadsM-280-Streptavidin, Dynal) were added and further incubated under gentleagitation for 30 minutes.

scFv phage that did not specifically bind to the target antigen wereeliminated by washing steps with PBS/0.1% BSA. For that purpose thebiotinylated antigen—streptavidin bead complexes (with the potentialscFv binders) were collected via a magnet and resuspended in 1 mL of thewashing solution (one washing step). This washing procedure was repeatedup to four times. After washing, binding entities were eluted by usingHCl-glycine pH 2.2 and after neutralization with 2 M Tris pH 12, theeluate was used for infection of a fresh uninfected E. coli XL1 Blueculture.

To elute remaining high binding entities the beads were resuspendeddirectly in 200 μL of a fresh E. coli XL1 blue culture (OD600≥0.5) andincubated for 10 minutes under gentle agitation. Both cultures were thenmixed and cells successfully transduced with a phagemid copy, encoding ahuman scFv-fragment, were again selected for carbenicillin resistanceand subsequently infected with VCMS 13 helper phage to start the secondround of antibody display and in vitro selection.

A total of 4 rounds of selections were carried out for the twoantibodies. Antigen concentrations were decreased during selection tothe final concentrations as follows:

1. round 100 nM 2. round  10 nM 3. round  10 nM 4. round  10 nM

Plasmid DNA from E. coli cultures was isolated corresponding to 3 and 4rounds of panning.

For the production of soluble scFv-protein the VH-VL-DNA fragments wereexcised from the plasmids (XhoI-SpeI), and cloned via the samerestriction sites in the plasmid pComb3H5BFlag/His (w/o additional phageproteins required for phage infection). After ligation each pool(different rounds of panning) of plasmid DNA was transformed into 100 μLheat shock competent E. coli TG1 and plated on carbenicillin LB-agar.Single colonies were picked and inoculated into 120 μL of LB carb (50μg/mL) 1% glucose in 96-well plates (Greiner). The wells were sealedwith a semipermeable membrane (Greiner) and the plates were incubatedovernight at 37° C. in a shaking incubator (master plate). Then 10 μL ofthe master plate cultures were transferred into a second 96 well plate(working plate) containing 90 μL LB carb (50 μg/mL) 0.1% glucose perwell. After incubation for 4 h in a 37° C. shaking incubator, scFvproduction was induced by adding 20 μL LB carb 6 mM IPTG to each well.After another incubation step overnight at 30° C. with shaking, cellswere lysed in a 1 h incubation at room temperature with 40 μL lysisbuffer (400 mM boric acid, 320 mM NaCl, 4 mM EDTA pH 8, 2.5 mg/mLlysozyme). Residual cells and cell debris were separated bycentrifugation for 12 minutes at 1,900×g (Hettich).

The supernatants containing scFv molecules were then tested for bindingin ELISA assays.

Detection of scFv-fragments bound to immobilized rhGM-CSF antigen(Leukine) was carried out using an anti-flag M2 (1 μg/mL PBS/1% BSA)detected with horseradish peroxidase-conjugated goat anti mouse Fab2specific polyclonal antibody (Dianova, 1 μg/mL PBS/1% BSA). The signalwas developed by adding ABTS substrate solution and detected at awavelength of 405 nm.

Of approximately 100 clones tested after the third selection round, 12clones showed strong binding to rhGM-CSF. Of approximately 160 clonestested after the fourth round over 80% of the lysates showed strongELISA signals as compared to PBS as a negative control on therecombinant antigen. Results from representative clones are depicted inFIG. 4, in which these representative clones are arrayed along thex-axis, and absorbance intensity is indicated on the y-axis. As can beseen from FIG. 4, the PBS negative control (second from right on thex-axis) showed no appreciable binding, whereas representative scFvclones scFv A, scFv 3035, scFv 3039, scFv 3080 and scFv 5-306 showeddifferent degrees of binding strength by ELISA.

All lysates were tested without rhGM-CSF in parallel experiments forunspecific binding to the blocking agent. No significant detectablesignal could be observed, indicating the specificity of the binding tothe rhGM-CSF.

The DNA sequences of more than 13 ELISA-positive scFv clones weredetermined In total, six different sequences were identified. Allsequences were of human origin and were closely related to the humangermline sequence VH-1 1-O2.

Example 2.3.5 Characterization of Human scFv Constructs Containing HumanVL and VH Regions Example 2.3.5.1 Large Scale Production andPurification of scFv Leads Constructs Produced by the Method Describedin Example 3

The scFv leads were isolated and purified as described in Example2.2.6.3.

Example 2.3.5.2 Kinetic Binding Analysis of scFv Leads by SurfacePlasmon Resonance (SPR)

The aim of the experiment is the in-depth characterization of the scFvleads. Binding kinetics (kd and ka) of the scFv leads were measured byinjecting 10 μL of purified protein in dilution series ranging from 10μg/mL to 1 pg/mL purified scFv and monitoring the dissociation at 25° C.for 100 sec. Protein was buffered in HBS-EP (0.01 M HEPES, pH 7.4, 0.15M NaCl, 3 mM EDTA, 0.005% surfactant P20). The data were fitted usingBIAevalution™ software determining the rate constant for dissociationand association kinetics with a 1:1 Langmuir binding equation (Formulae1 and 2), where A is the concentration of injected analyte and B is theconcentration of ligand.dB/dt=−(ka*[A]*[B]−kd*[AB])  (1)dAB/dt=−(ka*[A]*[B]−kd*[AB])  (2)

Kinetic binding curves were determined using up to 8 concentrations ofeach scFv lead analyzed. The independent fitting of the raw dataresulted in dissociation and association rate constants that were usedto calculate the equilibrium dissociation constant (KD, the results areshown in Table 1).

TABLE 1 IC50 ka [1/Ms] kd [1/s] KD [M] [nM] 3035 1.6 × 10⁵ ± 1.1 × 10⁵1.5 × 10⁻³ ± 0.4 × 10⁻³ 0.9 × 10⁻⁸ 3.2 3039 0.6 × 10⁴ ± 0.4 × 10⁴ 0.9 ×10⁻⁴ ± 0.1 × 10⁻⁴ 1.7 × 10⁻⁶ 130.5 scFvA 1.7 × 10⁶ ± 1.1 × 10⁶ 1.6 ×10⁻³ ± 0.2 × 10⁻³ 1.2 × 10⁻⁹ 2.6 3080 1.0 × 10⁵ ± 0.5 × 10⁵ 3.5 × 10⁻³ ±0.2 × 10⁻³ 3.5 × 10⁻⁸ 19.1

2.3.5.3: Inhibition of rhGM-CSF Dependent Proliferation of TF-1 Cells byscFv Leads

After confirming that the strength of specific binding was preserved inthe scFv leads, the aim of this experiment was to assess the specificityof the interaction of the scFv lead with the antigen rhGM-CSF. Theinhibition of the biological function of the antigen rhGM-CSF by bindingof the scFv was characterized in a TF-1 proliferation-inhibitionexperiment.

TF-1 proliferation-inhibition experiments were performed as describedabove. Cells were resuspended at a final concentration of 1×10 exp5cells/mL in RPMI 1640, 10% FCS and 90 μL cell suspension per well wereused (0.9×10 exp4 cells/well). A final concentration of 0.3 ng/mLrhGM-CSF was used to stimulate the proliferation of the TF-1 cells. Forneutralization of rhGM-CSF dependent proliferation purified scFv in1×PBS was added in a dilution series with final protein concentrationsranging from 100 μg/mL to 10 pg/mL. 10 μL of dialyzed and sterilefiltered protein solution (0.22 μm filter) was added to 100 μL TF-1 andrhGM-CSF solution. The samples were incubated at 37° C. at 5% CO₂ for 72h. After 72 h the proliferative status of the TF-1 cells was determinedadding WST-1 and monitoring the colorimetric change with an ELISA readerat 450 nm (FIG. 5). As can be seen in FIG. 5, the human GM-CSFneutralizing activity is clearly demonstrated. ScFv A displayed thestrongest neutralizing activity.

Example 2.4 Optimizing the Binding Characteristics of the Selected scFvs

It was contemplated that the biological activity of a neutralizing agentfor a monomeric ligand may be improved or even optimized by increasingthe binding strength between neutralizer and ligand, in particular byincreasing the off-rate of the neutralizer.

This can preferably be achieved by mutating the sequence of therespective VH and VL region in a random fashion by (i) inserting one ormore mutations randomly throughout the whole sequence or by (ii)inserting single mutations or multiple contiguous mutations (e.gstretches of five, six, seven, eight, nine or ten amino acids) intoregions of the scFv that have a high probability of interacting with theantigen. The respective mutants must then be characterized for anyincrease in activity or, prior to characterization, must be enriched forpreferential qualities (e.g. stronger binding) via suitable selectionmethods (i.e. phage display).

Example 2.4.1 Increasing the Affinity by Mutating the VH CDR3 in One orMore Positions

To improve the binding characteristics of an antibody fragment, forexample an scFv molecule, by single point mutations or short amino acidstretches, amino acid residues must be targeted which have a very highprobability of interacting with the respective antigen. With thisapproach, it is not necessary to screen more than only a limited numberof mutants without reducing the probability of success. The heavy chainCDR3 of an antibody or fragment thereof usually contributes strongly tothe overall binding of an antigen by this antibody or antibody fragment.It was therefore contemplated that a promising way of increasing thebinding affinity of an antibody or antibody fragment may be to mutatethe nucleotide sequence coding for VH CDR3.

A variety of different methodologies exists for performing such targetedrandom mutagenesis, some of which are described in the following interms of how the binding affinity of scFv molecules described above maybe increased:

-   -   A) To target the VH CDR3 a suitable restriction site must be        introduced into the nucleotide sequence within the VH CDR3,        preferentially by gene synthesis of the whole VH region with a        modified CDR3 nucleotide sequence by keeping the original amino        acid sequence (Entelechon, Germany). Via cleavage by the        respective restriction enzyme and adding S1 nuclease/Klenow DNA        polymerase I and dGTP followed by a mutant oligomer duplex,        targeted random mutagenesis in one or more amino acid positions        may be performed according to Matteucci and Heyneker, Nucleic        Acids Research 11: 3113 ff (1983). The mutagenized VHs are        subsequently combined with the respective VL (via a suitable        linker) in a suitable scFv expression vector and transformed        into E. coli cells. Single colonies expressing the variant scFvs        can then be picked and screened for improved scFvs as described        for screening and characterization of scFv hits and leads in the        previous examples.    -   B) An alternative method is the oligonucleotide-mediated        mutagenesis by the double primer method as described in detail        in Sambrook, Fritsch, Maniatis (1989) “A laboratory manual”. In        essence the VH region is cloned into an M13-based vector and        single stranded plasmid is isolated. A primer able to hybridize        to the single stranded plasmid template containing a randomized        sequence is annealed and extended. After propagation of the        respective plasmid pool in E. coli, the mutated VHs can be        harvested from the pool of plasmids and combined with the        original VL (via a suitable linker) in a suitable scFv        expression vector and transformed into E. coli cells. Single        colonies expressing the variant scFvs are picked and screened        for improved scFvs as described for screening and        characterization of scFv hits and leads in the previous        examples.    -   C) Yet another alternative is to mutate up to six or even more        contiguous amino acids. To this end, a deletion variant of the        VH nucleotide sequence may be constructed having a deleted CDR3        and FR4. This construct is used as a template for a one- or        two-step PCR amplification, in which a suitable 5′-primer        (hybridizing to the 5′ end of the VH sequence and adding a        suitable cloning site) is combined with a set of 3′-primers that        anneal at the 3′ end of the FR3 region as template and add a        CDR3 and FR4 region (with a suitable restriction site) to the        amplification fragment. This set of 3′-primers contains a        sequence of one or more triplets to insert random codons within        the CDR3 sequence. This pool of VH regions containing randomized        CDR3 regions may then be subsequently combined with the        respective VL (via a suitable linker) in a suitable scFv        expression vector and transformed into E. coli cells. Single        colonies expressing the variant scFvs are then picked and        screened for improved scFvs as described for screening and        characterization of scFv hits and leads in the previous        examples.

Respective pools of mutated scFvs that have a higher diversity (as canbe easily screened) can be cloned into a suitable phage display vectorand improved scFvs may then be selected by phage display on the antigenof interest preferentially under conditions of decreasing antigenconcentrations to select for higher affinities. Phage display selectionsare carried out according to standard protocols as described elsewhereherein. Any of the above methods A to C may be combined or performed inrepeated cycles to further improve and optimize already modified scFvs.

Example 2.4.2 Increasing the Affinity by Mutating the V-Regions RandomlyThroughout the Whole Sequence

Instead of mutating specific sites of the scFv that have a highprobability of interacting with the respective antigen, a more pragmaticapproach may be carried out by introducing point mutations throughoutthe entire VH and/or VL sequence and then screening for optimized scFvsor selecting and screening for optimized scFvs. By way of example, theVH and/or VL sequence may be mutagenized by using E. coli mutatorstrains (as described in Low et al. 260: 359 ff J Mol Biol (1996)) ormisincorporation of nucleotides by DNA polymerases as described indetail in Sambrook, Fritsch, Maniatis (1989) “A laboratory manual”.Cloning, expression and selection of optimized variants of scFvmolecules can be carried out by phage display or by the frequently usedribosome display technology (as described in EP 0 975 748 A1). Optimizedversions are expressed in suitable vector/E. coli systems to screen forimproved scFv candidates.

Suitable methodology as described above under Example 2.4 was applied tooptimize a representative scFv lead (scFv A), resulting in a class ofmonoclonal human anti-GM-CSF neutralizing antibody fragments representedby scFv molecules B-N. The characteristics of these scFv molecules willbe elucidated and described further in the following examples. Thegeneration of monoclonal IgG molecules from the selected scFv moleculesis described in the following example.

Example 3 Cloning and Eukaryotic Expression of Monoclonal Antibodiesfrom the Selected scFvs

Although bacteria are known to express functional Fab fragments, theyare usually not capable of producing complete functionalimmunoglobulins. For the production of complete functional antibodies,mammalian cells must be used and therefore the VL region of scFv 5-306and different VH regions of scFv molecules selected in the previousexamples were subcloned into mammalian expression vectors (especially VHregions of scFv A and scFv B).

Example 3.1 Cloning of the Human Light Chain Based on scFv 5-306

To generate suitable terminal restriction sites, the DNA fragmentencoding the VL region of scFv 5-306 was reamplified by PCR, resultingin Vkappa fragments with a Bsu36I-site at the 5′-end and a Xho I-site atthe 3′-end. This fragment was then subcloned into the plasmid BSPOLL byBsu36I and XhoI using the 5′-primer (5′-ACGTCACCTTAGGTGTCCACTCCGATATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGC-3′) and the 3′-primer(5′-CATGCACTCGAGCTTGGTCCCTCCGCCGAAAG-3′), thus adding a mammalian leadersequence and a human Ckappa constant region and verified by sequencing.Utilizing EcoRI and SalI, 5-306 VL-Ckappa DNA was excised from BSPOLLand subcloned into the eukaryotic expression vector pEF-ADA derived fromthe expression vector pEF-DHFR (Mack et al. (1995) Proc. Natl. Acad.Sci. USA. 92, 7021-5) by replacing the cDNA encoding murinedihydrofolate reductase (DHFR) with that encoding murine adenosinedeaminase (ADA).

Example 3.2 Cloning of Human Heavy Chain Variable Domains

From different human VH regions selected in the previous examples(especially VH regions of scFv A and scFv B), the variable region wasreamplified by PCR, generating Bsu36I restriction sites at both ends.For all constructs the combination of two primers was used: 5′-primerVH-Bsu36I (5′-ACGTCACCTTAGGTGTCCACTCCCAGGTGCAGCTGGT CCAGTCTGGGGCTGAGGTGAAGAAGC-3′) and 3′-primer (5′-ACGTCACCTGAGGAGACGGTGACCATTGTCCCTTG-3′). The resulting DNA-fragments were then subcloned using theserestriction sites into the eukaryotic expression vector pEF-DHFR alreadycontaining a eukaryotic leader sequence and a DNA-fragment encoding thehuman IgG1 heavy chain constant region. The heavy chain variable regionswere thus inserted between the leader and the heavy chain constantregion. The correct sequences of the variable regions were confirmed bysequencing.

Example 3.3 Conversion of scFv Fragments into Full Human IgGs

Plasmid encoding for the light chain (VL 5-306/Ckappa) and plasmidencoding for one heavy chain (VH/human IgG1 constant region) werecotransfected into HEK cells according to standard protocols fortransient protein expression and the cells were cultured to allow theexpression and production of the immunoglobulins into the culturemedium. In this manner, IgG A, deriving from scFv A and IgG B, derivingfrom scFv B were produced. After the respective production period, thesupernatants were harvested and the human immunoglobulins were isolatedvia Protein A chromatography according to standard protocols for thepurification of immunoglobulins. Purified immunoglobulins were then usedfor further characterization experiments.

Example 3.4 Reconversion of IgGs Specifities into scFv Fragments

VH regions from the IgG constructs (IgG A and IgG B, as described above)were recloned into a suitable scFv expression vector according tostandard protocols and were operatively coupled via a flexible linker tothe VL region stemming from the human light chain of Example 3.1. Theseconstructs were produced solubly in the periplasm of E. coli asdescribed above. The characterization of these scFvs (scFv O, derivedfrom IgG A, and scFv P, derived from IgG B) is described in thefollowing examples.

Example 4 Evaluation of Binding Specificity of a Human Monoclonalanti-GM-CSF Antibody for Primate and Human GM-CSF

The aim of this experiment was to show that an antibody obtained as setout above binds specifically to GM-CSF. Therefore the binding of such anantibody to different recombinant human (“rh”) colony-stimulatingfactors (rhG-CSF and rhM-CSF, Strathmann) was compared to the sameantibody's binding to rhGM-CSF by ELISA.

50 μL of the particular antigen (1 μg/mL in PBS) were coated onto anELISA plate (Nunc, Maxisorp) for 1 h at room temperature. After washing3 times with PBS/0.05% Tween 20 the wells were blocked with 200 μLPBS/3% non-fat dry milk powder per well for 1.5 h at room temperaturefollowed by washing 3 times with PBS/0.05% Tween 20. 50 μL/well of aseries of human antibodies (for example IgG A and IgG B), each withidentical light chains of sequence according to SEQ ID NO. 34 but withdifferent heavy chains according to SEQ ID NOs. 35-48 were added in adilution series ranging from 1 μg/mL to 0.5 ng/mL (in PBS/0.05% Tween20/3% non-fat dry milk powder) and incubated for 1 h. After 3 washeswith PBS/0.05% Tween 20, bound antibody was detected using 50 μL of ahorseradish-peroxidase-conjugated goat anti-human IgG antibody (Dianova;1:1000 diluted in PBS/0.05% Tween 20/3% non-fat dry milk powder). Thesignal was developed by addition of 50 μL/well ABTS solution (Roche) andabsorption was measured at 405 nm using a wavelength of 450 nm as areference.

Commercially available rabbit antibodies (Strathmann Biotech AG)specific for rhM-CSF and rhG-CSF, respectively, were used as positivecontrols for binding of these antigens, said binding being detected withan alkaline phosphatase-conjugated goat anti-rabbit antibody. The signalwas developed with 50 μL/well pNpp-solution (Sigma) and absorption wasmeasured at 405 nm using a wavelength of 450 nm as a reference.

The results are shown in FIGS. 6A, 6B and 6C for two representativehuman antibodies, IgG A and IgG B.

As can be seen in FIG. 6A, increasing concentration of titrated antibodyled to an increase in absorption, indicating good binding to rhGM-CSFfor both representative antibodies IgG A and B. FIG. 6B shows theresults of the same two representative antibodies binding to rhM-CSF. Ascan be seen in this figure, increasing concentrations of a rabbitanti-rhM-CSF antibody led to increasing absorption, i.e. increasingbinding of this control antibody (solid dots), whereas the tworepresentative antibodies described above (solid squares and solidtriangles) are superimposed as a continuing baseline absorbance whichdoes not increase with increasing test antibody concentration. Acompletely analogous result is seen for both control antibody as well asrepresentative test IgGs A and B in FIG. 6C, showing the results ofbinding to rhG-CSF.

Taken together, the data shown in FIGS. 6A, 6B and 6C indicate that thetwo representative test antibodies IgGs A and B specifically bind torhGM-CSF, but not to other colony stimulating factors such as M-CSF andG-CSF. Such antigen binding specificity is important for a promisingantibody therapeutic agent.

Example 5 Characterization of Binding Data for Human MonoclonalAnti-GM-CSF Antibodies and Fragments Thereof

It was desired to generate a qualitative ranking of various membersidentified as positive binders of rhGM-CSF by ELISA as described abovein Example 2. The ranking was intended to reflect kinetic (off-rate) andequilibrium (affinity) parameters of various representative antibodybinders so identified. To this end, surface plasmon resonance (SPR) wasperformed on the BIAcore™ 2000 apparatus, Biacore AB (Uppsala, Sweden)with a flow rate of 5 μL/min and HBS-EP (0.01 M HEPES, pH 7.4, 0.15 MNaCl, 3 mM EDTA, 0.005% surfactant P20) as running buffer at 25° C.Recombinant human GM-CSF (Leukine, Berlex, hereinafter alternatelyreferred to as “the antigen” or “rhGM-CSF”) produced in yeast wasimmobilized onto flow cells 2-4 on a CM5 sensor chip. The chip surfacewas activated by injecting 80 μL of 0.1 M sodium-hydroxysuccinimide, 0.4M N-ethyl-N′(3-dimethyl aminepropyl)-carbodiimide (NHS/EDC). The antigenwas coupled by manual injection of 10 μg/mL rhGM-CSF in 0.01 Msodium-acetate, pH 4.7. Different densities of antigen were immobilizedon flow cells 2-4 adjusting the amount of manual injection times. Flowcell 1 was left unmodified while flow cell 2 was coated with the highestpossible density of rhGM-CSF (800 RU). Flow cell 3 was coated with 50%of the amount of antigen immobilized on flow cell 2 and flow cell 4 wascoated with lowest density of rhGM-CSF (typically 10%). The activatedsurface of the sensor chip was blocked by injecting 85 μL of 1 Methanolamine and the chip was left to equilibrate overnight at aconstant flow rate of 5 μL/min of HBS-EP.

Example 5.1 Qualitative Determination of Kinetic Binding Parameters(Off-Rate) for scFv Fragments of Human Monoclonal Anti-GM-CSF Antibodies

Biacore experiments were performed as set out in the precedingparagraph. Prior to the experiment eluted protein solutions of theperiplasmic preparation (“PPP”) were dialyzed against PBS at 25° C. for2 h and diluted 1:1 in HBS-EP. Binding kinetics of the members ofclaimed class were measured by injecting 10 μL of purified periplasmicprotein solution at 25° C. over the sensor chip. The non-specificbackground adsorption of protein to the unmodified sensor chip surface(FC1) was subtracted from the response signal in the rhGM-CSFimmobilized flow cells (FC2, FC3, FC4). The relative response signal(FC2-1, FC3-1, FC4-1) was determined and the specific dissociation ratewas monitored for 100 sec.

The results of these experiments are shown in FIG. 7A for a series ofrepresentative scFv fragments which had previously been identified aspositive rhGM-CSF binders in ELISA experiments. Representative scFvantibody fragments for which Biacore data is shown in FIG. 7A are asfollows: scFv A, scFv B, scFv C, scFv D, scFv E, scFv F, scFv G, scFv H,scFv I, scFv J, scFv K, scFv L, scFv M and scFv N.

Generally, in interpreting Biacore results, the amplitude of the bindingpeak (RUmax) directly correlates to the protein concentration in theinjected sample. The kinetic on-rate (ka) is concentration dependentand, due to varying concentrations of protein in the PPP, cannot be usedfor the qualitative ranking of the members of claimed class. The kineticoff-rate (kd) is protein concentration independent and characteristicfor the binding strength of the respective members of claimed class. Allidentified members of claimed class show specific binding to theimmobilized rhGM-CSF. The members of claimed class with the bestapparent off rate were identified and after further correlation of theSPR data with the inhibition data submitted for determination ofaffinity via equilibrium binding experiments on the BIAcore.

In examining FIG. 7A, then, one sees distinct peaks for each ofrepresentative scFv antibody fragments A-N, the upper portions of whicheach show a characteristic curvature which can be extrapolated to obtainan off-rate for the scFv fragment in question. Qualitatively, then, onecan conclude that each of the representative scFv fragments binds wellto human GM-CSF.

Example 5.2 Quantitative Determination of Equilibrium Binding Parameters(Affinity) for Certain Human Anti-GM-CSF Antibodies and scFv FragmentsThereof

Having established, qualitatively, in Example 5.1 that a number of scFvfragments of anti-GM-CSF antibodies which had previously tested positivefor GM-CSF binding by ELISA demonstrate reasonable kinetic off-rateswhen binding to human GM-CSF, it was then desired to obtain aquantitative representation of such binding for antibodies and fragmentsthereof by focusing on the equilibrium binding characteristics torecombinant human GM-CSF. As shown above in Example 4, specific bindingto the antigen—here rhGM-CSF—is one of the characteristic and specificattributes of the class of anti-GM-CSF antibodies and fragments thereofas claimed herein.

Binding kinetics (the off-rate, kd, and the on-rate, ka) of certainrepresentative members of the class of human anti-GM-CSF antibodies andfragments thereof were measured by injecting 10 μL of purified protein(i.e. antibody or fragment thereof) in a dilution series ranging from 10μg/mL to 1 pg/mL purified protein and monitoring the dissociation at 25°C. for 100 sec. The purified protein was buffered in HBS-EP. The datawere fitted using BIAevalution™ software, determining the rate constantfor dissociation and association kinetics with a 1:1 Langmuir bindingequation (see Formulae 1 and 2 below), where A is the concentration ofinjected purified protein analyte and B[0] is Rmax:dB/dt=−(ka*[A]*[B]−kd*[AB])  (Formula 1)dAB/dt=−(ka*[A]*[B]−kd*[AB])  (Formula 2)

Kinetic binding curves were determined with up to 8 concentrations ofeach representative human anti-GM-CSF antibody or fragment thereofanalyzed. The independent fitting of the raw data resulted indissociation and association rate constants that were used to calculatethe equilibrium dissociation constant (KD). The results obtained foreach representative human anti-GM-CSF antibody or fragment thereof areshown in FIGS. 7B-I. Specifically, FIG. 7B shows the binding dataobtained for representative IgG B; FIG. 7C shows the binding dataobtained for representative IgG A; FIG. 7D shows the binding dataobtained for representative scFv C; FIG. 7E shows the binding dataobtained for representative scFv I; FIG. 7F shows the binding dataobtained for representative scFv B, FIG. 7G shows the binding dataobtained for representative scFv A; FIG. 7H shows the binding dataobtained for representative scFv E; and FIG. 7I shows the binding dataobtained for representative scFv D. The data are summarized below inTable 2.

TABLE 2 Quantitative affinity binding data for certain representativehuman anti-GM-CSF antibodies and fragments thereof Antibody/ fragment Inthereof Figure ka (s⁻¹M⁻¹) kd (s⁻¹) KD (M) scFv A 7G 4.41exp5 ± 3.00exp51.84exp−3 ± 6.55exp−4 4.17exp−9 scFv B 7F 1.01exp6 ± 4.08exp5 8.07exp−4± 3.73exp−5 7.98exp−10 scFv E 7H 1.26exp5 ± 5.57exp4 2.55exp−4 ±8.12exp−5 2.03exp−9 scFv C 7D 1.73exp5 ± 8.23exp4 4.77exp−4 ± 1.91exp−42.76exp−9 scFv D 7I 7.60exp5 ± 6.70exp5 8.66exp−4 ± 2.13exp−4 1.14exp−9scFv I 7E 2.32exp6 ± 2.13exp5 3.47exp−4 ± 8.78exp−5 1.50exp−9 IgG A 7C2.09exp6 ± 1.32exp6 1.81exp−4 ± 8.77exp−5 8.70exp−11 IgG B 7B 3.63exp5 ±2.40exp5 1.68exp−5 ± 5.74exp−6 4.64exp−11

Example 6 Minimal Epitope Requirements for a Certain RepresentativeFragment of a Human Anti-GM-CSF Antibody

It was desired to determine the epitope bound by human anti-GM-CSFantibodies and fragments thereof as described and claimed herein. Tothis end, a peptide spotting (“pepspot”) analysis was performed usingscFv A as a representative member of this class of molecules and humanGM-CSF as antigen.

Generally, a pepspot experiment is performed as follows. Overlapping13mer peptides derived from the amino acid sequence of hGM-CSF (for theGM-CSF sequence of humans and certain other primates, see hereinabove aswell as FIG. 8A, as well as SEQ ID NOs: 49-51) were covalently linked toa Whatman 50 cellulose membrane by the C-terminus while the acetylatedN-terminus remained free. The individual 13mer peptides generated (byJPT Peptide Technologies GmbH) are shown below in Table 3. The length ofthe overlapping sequence of any two respective peptides was set to be 11amino acids. According to the manufacturer's protocol the membrane wasrinsed with absolute EtOH for 1 min, followed by washing with TBS andblocking with TBS/3% BSA overnight. As with every subsequent incubationand washing, the blocking was carried out at room temperature. Afterwashing 3 times with TBS/0.05% Tween 20 for 10 min the membrane wasincubated with 1 μg/mL scFv A in TBS/3% BSA for 2.5 h followed bywashing carried out as before. Detection of the scFv was accomplished byusing an anti-Penta-His antibody (Qiagen, 0.2 μg/mL in TBS/3% BSA) andfollowed by a horseradish-peroxidase-conjugated goat-anti-mouse IgG(Fc-gamma-specific) antibody (Dianova, 1:10.000 in TBS/3% BSA) theincubation with each of these respective antibodies being performed for1 h. After washing 3 times with TBS/0.05% Tween 20 for 10 min the signalwas developed by enhanced chemiluminescence (SuperSignalWest PicoLuminol/Enhancer Solution and SuperSignalWest Pico StablePeroxideSolution; Pierce) and exposition to a BioMax Film (Kodak).

Strong binding signals of scFv A to stretches of human GM-CSF weredetected on a stretch of peptide-spots between spot A and B as well ason the spot C (See Table 3 below, and FIG. 8B). As can be seen in FIG.8B, binding to other spots seemed to be of a lower strength. The stretchof spots spanning points A and B corresponds to amino acid residues15-35. All 13mer peptides making up this region contain one minimalamino acid stretch of amino acids 23-27 (RRLLN). Spot C corresponds toamino acid residues 65-72 (GLRGSLTKLKGPL) of human GM-CSF. Thesefindings implicate that scFv A likely recognizes a discontinous epitope.

In the secondary structure of human GM-CSF amino acids 15-35 aresituated in helix A while residues corresponding to spot C are part of aloop-structure located between helices C and D. A three-dimensionalmodel of folding of the molecule reveals close sterical proximity ofthese sites with respect to one another.

The minimal amino acid sequence motif in the peptides of spots A-Bcorresponds to residues 23-27 of human GM-CSF (RRLLN). An increasingsignal strength from spot A to B can be explained by the betteraccessibility of the RRLLN epitope in peptide corresponding to spot Bthan in the peptide corresponding to spot A. In Peptide A the epitope islocated directly at the C-terminus that is linked to the membrane whilein peptide B it is located at the more accessible N-terminus of thepeptide.

TABLE 3 Sequences of overlapping 13mer peptides immobilized on the cellulose membrane.  1. APARSPSPSTQPW 2. ARSPSPSTQPWEH  3. SPSPSTQPWEHVN  4. SPSTQPWEHVNAI  5. STQPWEHVNAIQE 6. QPWEHVNAIQEAR  7. WEHVNAIQEARRL  

 

13. LLNLSRDTAAEMN 14. NLSRDTAAEMNET 15. SRDTAAEMNETVE 16. DTAAEMNETVEVI17. AAEMNETVEVISE 18. EMNETVEVISEMF 19. NETVEVISEMFDL 20. TVEVISEMFDLQE21. EVISEMFDLQEPT 22. ISEMFDLQEPTSL 23. EMFDLQEPTSLQT 24. FDLQEPTSLQTRL25. LQEPTSLQTRLEL 26. EPTSLQTRLELYK 27. TSLQTRLELYKQG 28. LQTRLELYKQGLR29. TRLELYKQGLRGS 30. LELYKQGLRGSLT 31. LYKQGLRGSLTKL 32. KQGLRGSLTKLKG

34. RGSLTKLKGPLTM 35. SLTKLKGPLTMMA 36. TKLKGPLTMMASH 37. LKGPLTMMASHYK38. GPLTMMASHYKQH 39. LTMMASHYKQHSP 40. MMASHYKQHSPPT 41. ASHYKQHSPPTPE42. HYKQHSPPTPETS 43. KQHSPPTPETSSA 44. HSPPTPETSSATQ 45. PPTPETSSATQTI46. TPETSSATQTITF 47. ETSSATQTITFES 48. SSATQTITFESFK 49. ATQTITFESFKEN50. QTITFESFKENLK 51. ITFESFKENLKDF 52. FESFKENLKDFLL 53. SFKENLKDFLLVI54. KENLKDFLLVIPF 55. NLKDFLLVIPFDS 56. KDFLLVIPFDSWE 57. FLLVIPFDSWEPV58. LVIPFDSWEPVQE

Example 7 Neutralization Potency of Certain Human Anti-Human GM-CSFAntibodies/Antibody Fragments Example 7.1 Qualitative Evaluation ofNeutralization Potential of Certain Representative Human Anti-HumanGM-CSF Antibodies and Fragments Thereof

The aim of this experiment is to achieve qualitative information on theneutralizing activity of representative human anti-GM-CSF neutralizingantibodies and fragments thereof. To this end, the humanGM-CSF-dependant cell line TF-1 (DSMZ, ACC 334) was used. The rate ofproliferation of this cell line depends on the presence of human GM-CSF,so that measuring cell growth following incubation of cells with humanGM-CSF with and without an antibody suspected of havingGM-CSF-neutralizing activity may be used to determine whether suchneutralization activity in fact exists.

TF-1 cells were cultivated in RPMI 1640 medium (Gibco; L-glutamine,phenol-red free), 10% heat inactivated FCS in the presence of 2.5 ng/mLrhGM-CSF as described by the distributor (Deutsche Sammlung vonMikroorganismen and Zellkulturen GmbH, Braunschweig, Germany). Cellswere grown to a cell density of 0.5×10 exp6 cells/mL. For theproliferation assay TF-1 cells were harvested by centrifugation at 300×gfor 4 min and washed with 1×PBS (Dulbecco's, Gibco). Cells wereresuspended to a final concentration of 1×10 exp5 cells/mL in RPMI 1640,10% FCS and 90 μL cell suspension per Microtest flat bottom cell cultureplate well were used (0.9×10 exp4 cells/well). A final concentration of0.3 ng/mL rhGM-CSF was used to stimulate the proliferation of the TF-1cells. For neutralization of GM-CSF dependent proliferation purified PPPof representative fragments of a human anti-GM-CSF antibody weredialyzed against 1×PBS at 25° C. for 2h. 10 μl of dialyzed and sterilefiltered protein solution (0.22 μm filter) were added to 100 μl solutioncontaining TF-1 and rhGM-CSF.

After incubation for 72 h at 37° C. at 5% CO₂ the proliferative statusof the TF-1 cells was determined with a colorimetric assay based on thecleavage of tetrazolium salts (WST-1, Roche) by mitochondrialdehydrogenase in viable cells. The formazan dye formed by metabolicallyactive cells was quantitated by measuring its absorbance with an ELISAreader at 450 nm.

The inhibition of the human GM-CSF-dependant proliferation of TF-1 cellsby the tested representative fragments of human anti-human GM-CSFantibody fragments was varying in strength (FIG. 9). While two suchfragments did not have a neutralizing effect (scFv F and scFv L); fiveconstructs (scFv J, scFv K, scFv M, scFv N, and scFv H) showedintermediate inhibition and seven constructs (scFv B, scFv I, scFv E,scFv D, scFv G, scFv C, scFv A) showed strong inhibition of the GM-CSFdependant proliferation of TF-1 cells. The lack or lower degree ofneutralizing effect could be due to a lower expression level of theparticular representative scFv or to a less stable complex formedbetween a particular representative scFv and rhGM-CSF over theincubation period of 72 h at 37° C.

Example 7.2 Quantitative Evaluation of Neutralization Potential ofCertain Representative Human Anti-Human GM-CSF Antibodies and FragmentsThereof, as Measured by Cell Proliferation

Selected representative scFv molecules shown above to exhibit stronginhibition of TF-1 proliferation were then subjected to a quantitativeanalysis of neutralizing efficacy. To this end, the same humanGM-CSF-dependant cell line TF-1 (DSMZ ACC 334) was used. TF-1 cells werecultivated and prepared for the proliferation assay as described indetail in Example 7.1 above. A final concentration of 0.3 ng/mL rhGM-CSFwas used to stimulate the proliferation of the TF-1 cells. Forneutralization of GM-CSF-dependent proliferation 10 μl of purifiedsamples of representative human anti-human GM-CSF neutralizingmonoclonal antibodies or fragments thereof were added to a solutioncontaining 100 μl TF-1 and rhGM-CSF in a dilution series. Final proteinconcentrations ranged from 10 μg/ml to 10 pg/ml.

Samples were incubated at 37° C. at 5% CO₂ for 72 h. After 72 h theproliferative status of the TF-1 cells was determined as described inExample 7.1 above. The data were fitted for half maximal inhibition ofproliferation (IC₅₀) using the non-linear regression curve fit of thePrism software.

The clear GM-CSF neutralizing effect seen in the qualitativeproliferation-inhibition experiment described in Example 7.1 above couldbe confirmed and quantified. All tested scFv fragments of humananti-human GM-CSF monoclonal neutralizing antibodies displayed a halfmaximal inhibition constant (IC₅₀) in the nanomolar range in thisproliferation-inhibition experiment. A clear ranking in neutralizingefficacy could be established, as is seen in FIG. 10A.

The tested human anti-human GM-CSF monoclonal neutralizing IgGantibodies display a significantly higher neutralizing efficacy thantheir scFv counterparts. The half maximal inhibition constant of the IgGmolecules generated in this experiment was in the sub-nanomolar range.As can be seen in FIG. 10B, the IC₅₀ evaluated for IgG A was 0.9 nM andIgG B had an IC50 of 0.3 nM.

In order to check whether the scFv antibody fragments generated fromIgGs A and B (scFvs O and P, respectively) quantitatively correspond intheir neutralization potential to scFvs A and B, analogous TF-1neutralization assays were performed as described above except usingscFvs O and P as test molecules. The results are shown in FIGS. 10C and10D for scFvs P and O, respectively. As can be seen from FIG. 10D, scFvO has the same neutralization potential as scFv A, showing thatreconversion from IgG back to scFv format is possible without loss ofbiological activity.

Example 7.3 Quantitative Evaluation of Neutralization Potential ofCertain Representative Human Anti-rhGM-CSF Antibodies and FragmentsThereof, as Measured by Reduced IL-8 Production

This experiment was performed to quantify the neutralization activity ofrepresentative human anti-human GM-CSF antibodies and fragments thereofby measuring GM-CSF-dependent IL-8 production by U-937 cells. The GM-CSFantigen used in the foregoing experiments was rhGM-CSF. The monocyticU-937 cells were cultivated in RPMI 1640 medium Gibco (L-glutamine,phenol-red free) supplemented with 10% heat inactivated FCS as describedby the distributor (Deutsche Sammlung von Mikroorganismen andZellkulturen GmbH, Braunschweig, Germany). Cells were grown to a celldensity of 1×10 exp6 cells/mL.

In performing the inhibition assay based on measurement of IL-8production, cells were harvested by centrifugation at 300×g for 4 minand resuspended to a final concentration of 1×10 exp6 cells/mL in RPMI1640, 10% FCS. 1.8×10 exp5 cells/well (180 μL cell suspension) wereseeded per Microtest flat bottom cell culture plate well. A finalconcentration of 1 ng/mL rhGM-CSF was used to stimulate IL-8 productionby the U-937 cells. 20 μl of purified scFv or IgG was added to 180 μlU937 cells and rhGM-CSF solution in a dilution series resulting in finalprotein concentrations ranging from 10 μg/mL to 10 pg/mL

After incubation for 18 h at 37° C. and 5% CO₂ cells were spun down bycentrifugation of culture plates for 2 min at 600×g. Culturesupernatants were harvested by pipetting to a new plate and wereanalyzed to determine the concentration of IL-8 therein using the OptEIAHuman IL-8 ELISA Set (Becton Dickenson and Company).

ELISA detection was carried out according to the manufacturer'sinstructions. In brief, 50 μL of capture antibody diluted in 0.1 Msodium carbonate, pH 9.5 were coated onto a microtest plate over nightat 4° C. After washing 3 times with PBS/0.05% Tween 20 the wells wereblocked with 200 μL PBS/10% FCS per well for 1 h at room temperaturefollowed by washing 3 times with PBS/0.05% Tween 20. Then 50 μL of theculture supernatant samples were added to the wells and incubated for 2h at room temperature. For later quantification of the IL-8concentration a serial dilution of the IL-8 standard provided by themanufacturer was carried along through the procedure.

After washing 5 times with PBS/0.05% Tween 20 detection was carried outusing 50 μL of the Working Detector (Detection Ab+Av-HRP) provided inthe OptEIA Human IL-8 ELISA Set. After a 1 h incubation at roomtemperature, wells were washed an additional 7 times. The signal wasdeveloped by adding OPD substrate solution (Sigma) and was detected at awavelength of 490 nm (using a reference wavelength of 620 nm).

An IL-8 standard curve was plotted for calibration and IL-8concentration in the culture supernatant samples was calculatedaccording to this calibration curve. The data were fitted for halfmaximal inhibition of IL-8 production (IC₅₀) using the non-linearregression curve fit of the Prism software.

All representative fragments of human anti-rhGM-CSF monoclonalneutralizing antibodies tested showed clear inhibition of the GM-CSFdependent IL-8 production of U-937 cells, as can be clearly seen by thedecrease in IL-8 concentration with increasing scFv concentration inFIG. 11. The ranking in neutralizing efficacy seen in this experiment isin accordance with the ranking obtained testing the same molecules fortheir neutralizing effect in the TF-1 proliferation-inhibitionexperiment described above.

It will be noted that the IC₅₀ values determined in this experiment arehigher as compared to those obtained for the same molecules in theprevious TF-1 proliferation experiment. This is due to the higher GM-CSFconcentration required for stimulation of IL-8 production by U-937 cellsthan required for stimulation of TF-1.

Example 7.4 Quantitative Evaluation of Neutralization Potential ofRepresentative Human Anti-Human GM-CSF Antibodies and Fragments hereofon Recombinant Macacan GM-CSF, as Measured by Cell Proliferation

The aim of this experiment was to show the neutralizing potency ofrepresentative human anti-human GM-CSF antibodies and fragments thereoffor GM-CSF from non-human primates of the Macaca family (“macGM-CSF”).

To show the neutralizing effect of selected scFv and IgG molecules onmacGM-CSF, a proliferation-inhibition experiment was performed accordingto the protocol described in Examples 7.1 and 7.2 using macGM-CSFinstead of hGM-CSF. Both hGM-CSF and macGM-CSF stimulate theproliferation of TF-1 cells with the same half maximal efficacy (EC₅₀).A final concentration of 3 ng/ml macGM-CSF was used to stimulate theproliferation of the TF-1 cells in the experiment testing the scFvmolecules and 0.3 ng/mL rhGM-CSF cells in the experiment testing IgG Bas a representative human anti-human GM-CSF antibody. In order toneutralize the proliferation of the TF-1 cells, 10 μl of purified humananti-human GM-CSF antibody or fragment thereof were added to 100 μl TF-1and macGM-CSF solution in a dilution series. Final proteinconcentrations ranged from 10 μg/ml to 10 pg/ml. Samples were incubatedat 37° C. at 5% CO₂ for 72 h. After 72 h the proliferative status of theTF-1 cells was determined as described in Examples 7.1 and 7.2. The datawere fitted for half maximal inhibition of proliferation (IC₅₀) usingthe non-linear regression curve fit of the Prism software.

As seen in FIG. 12A, certain representative human anti-human GM-CSFmonoclonal antibody fragments also exhibited a clear neutralizationpotential of mac GM-CSF (scFv B, scFv E, scFv C, scFv I, scFv A).Furthermore, as can be seen in FIG. 12B, increasing concentrations ofthe representative human anti-human GM-CSF monoclonal antibody IgG Bclearly led to a decrease in TF-1 proliferation, demonstrating thisantibody's neutralizing potential. Interestingly, the IC50 valuegenerated for IgG B in this experiment (0.3 nM) using mac GM-CSF forinduction of TF-1 cell proliferation is equal to the one generated inthe experiment using hGM-CSF, showing a clear cross-reactivity of IgG Bfor GM-CSF in these species.

Example 8 Cross-Reactivity of IgG B with GM-CSF from Various Species

The cross-reactivity of IgG B with GM-CSF from various non-human specieswas investigated to identify species suitable for later in vivo studies.In a first set of experiments, binding of IgG B to commerciallyavailable recombinant GM-CSF from human (Leukine®, Berlex), pig, dog,rat (R&D Systems, Wiesbaden, Germany) and mouse (Strathmann Biotech,Hamburg, Germany) was tested in an ELISA experiment. Specifically, anELISA-plate was coated with 1 μg/mL GM-CSF from the various speciesmentioned. IgG B was added in a dilution series and was detected using ahorseradish-peroxidase-conjugated anti-human IgG1 antibody. The ELISAwas developed by adding OPD o-phenylendiamine (“OPD”, yellow-orange whenreacted with peroxidase) substrate solution (Roche, Germany) andmeasured at 490 nm

As seen in FIG. 13, IgG B showed robust binding to recombinant humanGM-CSF, while GM-CSF from the other species tested was not recognized.Pig, dog, rat or mouse may therefore not be suitable species for in vivotesting. However, as seen above in Example 7.4, IgG B shows a markedcross-reactivity with macGM-CSF (from cynomolgous monkey, macacafascicularis), implying the suitability of at least one monkey speciesfrom the macacan family for in vivo studies of IgG B.

Example 9 Binding by IgG B to Differently Glycosylated Variants ofGM-CSF

The aim of this experiment was to determine the extent to which thebinding of IgG B to GM-CSF depends on the latter's glycosylationpattern. To this end, a dilution series of conditioned medium containingnatural hGM-CSF (human glycosylation), as well as recombinant hGM-CSFfrom E. coli (no glycosylation) and yeast (yeast glycosylation), as wellas recombinant macaque GM-CSF were tested for their potency to induceTF-1 proliferation.

Human glycosylated GM-CSF was obtained from the culture supernatant ofIL-1β-treated BEAS-2B cells (human lung cells obtained from ATCCCRL-9609). BEAS-2B cells were propagated in BEBM-Medium substituted withthe BEGM Bullet Kit (Cambrex, Verviers, Belgium) but cultured in RPMI1640, 10% FCS in the presence of 50 ng/mL IL-1β (Strathmann Biotech,Hamburg, Germany) for induction of GM-CSF production. After 48-hourincubation at 37° C., 5% CO₂ the culture supernatant was analyzed forits GM-CSF content using the OptEIA Human GM-CSF Elisa Set (BDBiosciences, Heidelberg, Germany) according to the manufacturer'sinstructions.

Recombinant hGM-CSF from E. coli was internally produced as set out inExample 1.1 of WO 2005/105844. Recombinant hGM-CSF from yeast wasobtained commercially under the trade name “Leukine” (Berlex, USA).Macaque GM-CSF was recombinantly produced in HEK293 cells.

A dilution series of conditioned medium containing natural hGM-CSF, aswell as recombinant hGM-CSF from E. coli and yeast, and macaque GM-CSFwere first tested for their potency to induce TF-1 proliferation. Allthree glycosylation variants of human GM-CSF and macaque GM-CSFexhibited very similar EC50 values for TF-1 activation. These were 10pg/mL for E. coli-produced hGM-CSF, 15 pg/mL for yeast-produced hGM-CSF,36 pg/mL for human lung cell-produced hGM-CSF, and 11 pg/mL for macaqueGM-CSF, respectively (FIG. 14A).

The neutralizing activity of IgG B was then determined in the presenceof 0.3 ng/mL recombinant hGM-CSF, or 0.2 ng/mL physiological hGM-CSF.After 72 hours, the proliferative status of TF-1 cells in the presenceof different IgG B concentrations was quantified by a colorimetricreaction (FIG. 14B).

Taken together, the data shown in FIG. 14 show that IgG B inhibitedGM-CSF-dependent proliferation of TF-1 cells at sub-nanomolarconcentrations apparently independent of the glycosylation pattern ofhuman GM-CSF. The glycosylation pattern of human GM-CSF therefore doesnot substantially influence the ability of IgG B to neutralize GM-CSFactivity.

Example 10 Effect of IgG B on Biological Activities of GM-CSF onEosinophils Example 10.1 Effect of IgG B on GM-CSF-Mediated EosinophilSurvival

One of the various biological activities of GM-CSF is prolongation ofeosinophilic and neutrophilic granulocyte survival. Because lunginflammatory diseases are associated with local accumulation ofeosinophils, which play a substantial role in maintaining inflammation,the efficacy of IgG B in inhibiting GM-CSF-mediated eosinophil survivalwas tested.

Eosinophils were isolated from peripheral blood of healthy donors bydepletion of CD16⁺ neutrophils from the granulocyte population obtainedby density gradient centrifugation and lysis of erythrocytes. Freshlyisolated peripheral blood eosinophils were seeded at a density of 5×10⁴cells/well in RPMI 1640/10% FCS and Pen/Strep in a 96-well flat bottommicrotest plate. GM-CSF was added in a dilution series ranging from 33ng/mL to 10 pg/mL to monitor the concentration-dependent eosinophilsurvival. To analyze the inhibiting potential of IgG B onGM-CSF-dependent eosinophil survival, the antibody was added in adilution series ranging from 10 μg/mL to 0.1 ng/mL. A finalconcentration of 0.1 ng/mL GM-CSF was used to effect eosinophilsurvival. After incubation for 72 h at 37° C., 5% CO₂ WST-1 reagent wasadded. The resulting colorimetric reaction corresponding to the portionof viable cells was quantified by measuring the absorbance at 450 nm Thedata were analyzed and fitted for half-maximal inhibition of survival(IC50) using the non-linear regression curve fit of the prism softwarepackage. As seen in FIG. 15A, a half-maximal effective dose (EC50) of0.02 ng/mL rhGM-CSF was determined As seen in FIG. 15B, a potentneutralizing effect of IgG B was seen with a half-maximal inhibition ofeosinophil survival at an antibody concentration of 0.13 nM.

These data indicate that IgG B is effective in inhibitingGM-CSF-dependent-eosinophil survival in a dose-dependent manner.

Example 10.2 Effect of IgG B on GM-CSF-Induced Eosinophil Activation

It was also desired to investigate the effect of IgG B on GM-CSF-inducedactivation of eosinophils. CD69 expression was found to be up-regulatedon peripheral eosinophils (CD16⁻) isolated from human blood followingstimulation for 20 h or 3 days with (a) 0.1 ng/mL GM-CSF or (b) 0.1ng/mL GM-CSF, IL-3 and IL-5, but not with (c) 0.1 ng/mL IL-3 and IL-5alone (FIG. 16A). Eosinophils cultured in the presence of medium aloneshowed no up-regulation of CD69. CD69 may therefore be taken as a markerfor eosinophil activation by GM-CSF, and the expression level of CD69was monitored as a measure of GM-CSF-dependent eosinophil activation. Atboth time points (20 h and 3 days), IgG B (10 μg/mL) almost completelyprevented GM-CSF-dependent activation of eosinophils, as seen by lack ofCD69 expression in flow cytometry.

Eosinophils were isolated as described above in Example 10.1 andcultivated at a density of 5×10⁵ cells/well in RPMI 1640/10% FCS andPen/Strep in a 48-well flat bottom microtest plate. Cells were incubatedwith medium alone or in the presence of 0.1 ng/mL GM-CSF alone ortogether with 0.1 ng/mL IL-3 or IL-5. 10 μg/mL IgGB were used forneutralization of GM-CSF. After an incubation of 1 or 3 days cells wereanalyzed for CD69 expression by flow cytometry.

CD69 detection by flow cytometry: Expression of CD69 on eosinophils wasdetermined on a FACS Calibur instrument (Becton Dickinson). 10⁵ cellswere incubated with 5 μL of a FITC-conjugated anti-human CD16 (clone3G8, BD Biosciences) and a PE-conjugated anti-human CD69 antibody (cloneFN50, BD Biosciences) each for 1 h at 4° C. As a negative controlirrelevant, isotype-matched FITC- and PE-conjugated antibodies wereused. After incubation, cells were washed twice with PBS, 1% FCS, 0.05%NaN₃ and resuspended in 250 μL PBS, 1% FCS, 0.05% NaN₃. Propidium iodidewas added to label dead cells to a final concentration of 1 μg/mLimmediately before FACS analysis. Data interpretation was done using theCellQuestPro software (BD Biosciences). Propidium iodide-positive (i.e.dead) cells were excluded from analysis of CD69 expression.

IgG B also reduced the percentage of live and activated eosinophils asmonitored by propidium iodide staining of CD16⁻/CD69⁺ cells in thepresence of 0.1 ng/mL GM-CSF. IgG B reduced the percentage of activatedcells from 35% to 8% after 1 day and from 43% to 3% after 3 days ofcultivation. In the presence of 0.1 ng/mL GM-CSF, IL-3 and IL-5, thepercentage of live and activated eosinophils was reduced from 32% to 8%and from 48% to 11% after 1 and 3 days, respectively. Even though theupregulation of CD69 was completely inhibited by IgG B, higher numbersof resting eosinophils (CD16⁻/CD69⁻) survived for 3 days in the presenceof 0.1 ng/mL GM-CSF, IL-3 and IL-5 as compared to cells incubated withmedium or GM-CSF alone (FIG. 16A, last column). The same was observedfor cells incubated in the presence of 0.1 ng/mL IL-3 plus IL-5.

In dose finding experiments, IgG B was added in dilution series toeosinophils cultured in the presence of 0.1 ng/mL GM-CSF (FIG. 16B). Aninhibitory effect of IgG B on CD69-dependent median fluorescenceintensity (MFI) was observed at a half-maximal concentration of 0.22 nMIgG B.

Taken together, these data indicate that IgG B is an effectiveneutralizer of GM-CSF activity in a biological context highly relevantfor inflammatory airway diseases, for example asthma.

Example 11 Preliminary Ex Vivo Toxicology Studies using IgG B

As explained above, neutralization of GM-CSF activity can betherapeutically advantageous in a number of disease settings. At thesame time, however, GM-CSF plays an important role in the normalfunction of the immune system in combating exogeneous pathogens, forexample as in phagocytosis by neutrophil granulocytes and monocytes.This natural function of neutrophils and monocytes should remainunaffected in the presence of therapeutic amounts of IgG B. Therefore weinvestigated two aspects of the phagocytic process: 1) ingestion ofbacteria (phagocytosis); and 2) oxidative burst activity (indicative forintracellular killing). These studies are detailed in the followingexamples.

Example 11.1 Ingestion of Bacteria (Phagocytosis)

Determination of granulocyte and monocyte phagocytic activity inheparinized whole blood was performed using the Phagotest Kit by Orpegen(Heidelberg, Germany). This test is based on the ingestion of opsonized,fluorescent-labelled E. coli by phagocytic cells. These cells can thenbe detected by green fluorescence in flow cytometry. 20 μlfluorescein-labelled opsonized E. coli were added to 100 μl ofheparinized whole blood and incubated at 37° C. Incubation at 0° C. wasperformed as a negative control. After 10 min the phagocytic process wasstopped by cooling samples on ice and addition of 100 μl Quenchingsolution (Orpegen). This solution allows discrimination of attachmentand internalization of bacteria by quenching FITC fluorescence ofsurface bound bacteria while fluorescence of internalized particlesremains unaffected. After three washing steps with 3 ml washing solution(Orpegen), erythrocytes were lysed. The remaining leucocytes were oncewashed with 3 ml Washing solution (Orpegen). After addition of 200 μlDNA staining solution, that allows exclusion of aggregated bacteria orcells, the cells were analyzed by flow cytometry. The percentage ofcells having performed phagocytosis was determined by means ofFITC-fluorescence.

To determine the influence of IgG B on phagocytosis, IgG B was added tothree identical blood samples to a final concentration of 10 μg/ml.These three samples were then allowed to incubate at 37° C. with IgG Bfor various amounts of time prior to addition of E. coli. E. coli wereadded to the first sample immediately, whereas E. coli were added to thesecond and third samples after 24 and 48 hours, respectively.

Results observed for granulocytes: Directly after blood was taken over98% of granulocytes ingested bacteria either in the presence or absenceof IgG B (FIG. 17A). After incubation of blood samples with IgG B for 24h a decrease to around 92% was determined without IgG B and to 90% inthe presence of IgG B (FIG. 17B). After 48 h 81% of the granulocyteswere phagocytosis positive in the absence and 89% in the presence of IgGB (FIG. 17C).

Results observed for monocytes: Irrespective of IgG B being present ornot 98% monocytes were phagocytosing directly after blood was taken(FIG. 18A). After 24 h pre-incubation with IgG B 90% of the monocyteswere positive (FIG. 18B). After 24 h pre-incubation without IgG B itwere 92% monocytes. After 48 h we found 81% of the monocytes without IgGB and 89% with IgG B phagocytosis positive (FIG. 18C).

Example 11.2 Oxidative Burst

Determination of granulocyte and monocyte oxidative burst activity inheparinized whole blood was performed using the Phagoburst Kit byOrpegen (Heidelberg, Germany). This assay allows determination of thepercentage of phagocytic cells which produce reactive oxidants byoxidation of the substrate dihydrorhodamine (DHR) 123 to the fluorescentR 123. Cells exhibiting oxidative burst activity can be identified inflow cytometry. Heparinized blood was incubated with different stimulito induce oxidative burst activity: phorbol 12-myristate 13-acetate(“PMA”) as a high stimulus; unlabelled, opsonized E. coli asintermediate stimulus and the chemotactic peptide N-formyl-MetLeuPhe(fMLP) as low stimulus. 100 μl whole blood was incubated with thesestimuli at 37° C. As a negative control incubation was performed withoutstimulation. After 10 min incubation DHR 123 substrate solution wasadded and incubated for another 10 min DHR 123 is converted to thefluorescent R 123 by oxidizing cells. After three washing steps with 3ml Washing solution (Orpegen), erythrocytes were lysed. The remainingleucocytes were once washed with 3 ml Washing solution (Orpegen). Afteraddition of 200 μl DNA staining solution, that allows exclusion ofaggregated bacteria or cells, the cells were analyzed by flow cytometry.

To determine the influence of IgG B on oxidative burst, IgG B was addedto three identical blood samples to a final concentration of 10 μg/ml.Each of these three samples was then divided into three aliquots andallowed to incubate at 37° C. for various amounts of time prior toaddition, to separate aliquots, of E. coli, fMLP or PMA. E. coli, fMLPor PMA were added to the three aliquots of the first sample immediately,whereas E. coli, fMLP or PMA were added to the three aliquots of thesecond and third samples after 24 and 48 hours, respectively. Parallelblood samples lacking IgG B were treated identically as above ascontrols. The results are shown below in Table 4, where “+” in thesecond column from the left indicates that IgG B is present in thesample aliquot tested, and “−” in the second column from the leftindicates the IgG B-free control.

TABLE 4 Effect of IgG B on oxidative burst behaviour of granulocytesPercent oxidizing granulocytes Results following stimulation with...shown Timepoint IgG B E. coli fMLP PMA in . . .  0 h − 97 9 99 FIG.17D + 94 10 99 24 h − 82 8 97 FIG. 17E + 80 10 95 48 h − 68 6 64 FIG.17F + 71 5 64

Similar results were obtained using monocytes instead of granulocytes.The experiment was performed analogously as described above, and theresults are shown below in Table 5, where “+” in the second column fromthe left indicates that IgG B is present in the sample aliquot tested,and “−” in the second column from the left indicates the IgG B-freecontrol.

TABLE 5 Effect of IgG B on oxidative burst behaviour of monocytesPercent oxidizing monocytes Results following stimulation with... shownTimepoint IgG B E. coli fMLP PMA in . . .  0 h − 62 0 79 FIG. 18D + 57 181 24 h − 30 4 35 FIG. 18E + 26 6 28 48 h − 29 4 17 FIG. 18F + 28 3 14

Overall it can therefore be concluded that the presence of IgG B atphysiologically relevant temperatures did not adversely affect thephagocytosis or oxidative killing of bacteria by either granulocytes ormonocytes. In an in vivo context, these results suggest, then, thattherapeutic administration of IgG B would not be expected to adverselyaffect the normal immune defenses of the patient.

The invention claimed is:
 1. A method of treating an inflammatorydisease, comprising administering an effective dose of a humanmonoclonal antibody or fragment thereof which specifically binds to andneutralizes primate GM-CSF, wherein the antibody or fragment thereofcomprises in its light chain variable region a CDR1 comprising an aminoacid sequence as set out in SEQ ID NO: 16, a CDR2 comprising an aminoacid sequence as set out in SEQ ID NO: 17 and a CDR3 comprising an aminoacid sequence as set out in SEQ ID NO: 18; and comprising in its heavychain variable region a CDR1 comprising an amino acid sequence as setout in SEQ ID NO: 14, a CDR2 comprising an amino acid sequence as setout in SEQ ID NO: 15 and a CDR3 comprising an amino acid sequence as setout in any of SEQ ID NOS: 1-13 or 56, wherein the inflammatory diseaseis rheumatoid arthritis (RA), asthma, or multiple sclerosis (MS).
 2. Themethod of claim 1, wherein the primate GM-CSF is a human GM-CSF.
 3. Themethod of claim 1, wherein the antibody or fragment thereof isadministered in combination with one or more anti-inflammatory agents.4. The method of claim 1, wherein the human monoclonal antibody is IgG.5. The method of claim 4, wherein the IgG is an IgG1 or IgG4.
 6. Themethod of claim 1, wherein the human monoclonal antibody or fragmentthereof comprises in its light chain variable region an amino acidsequence as set out in any of SEQ ID NOs: 19, 54 or
 55. 7. The method ofclaim 1, wherein the human monoclonal antibody or fragment thereofcomprises in its heavy chain variable region an amino acid sequence asset out in any of SEQ ID NOs: 20-33, 52 or
 53. 8. The method of claim 1,wherein the human monoclonal antibody or fragment thereof comprises alight chain amino acid sequence as set out in SEQ ID NO: 34 and a heavychain amino acid sequence as set out in any of SEQ ID NOs: 35-48.
 9. Themethod of claim 8, wherein the human monoclonal antibody or fragmentthereof comprises a light chain amino acid sequence as set out in SEQ IDNO: 34 and a heavy chain amino acid sequence as set out in SEQ ID NO:35.
 10. The method of claim 1, wherein the human monoclonal antibodyfragment is an scFv, an Fv, a diabody, a tandem diabody, a Fab, a Fab′,or a F(ab)₂.
 11. A method of treating an inflammatory disease,comprising administering an effective dose of a human monoclonalantibody or fragment thereof which specifically binds to and neutralizesprimate GM-CSF, wherein the antibody or fragment thereof comprises inits light chain variable region a CDR1 comprising an amino acid sequenceas set out in SEQ ID NO: 16, a CDR2 having an amino acid sequence as setout in SEQ ID NO: 17, and a CDR3 having an amino acid sequence as setout in SEQ ID NO: 18, and comprises in its heavy chain variable region aCDR1 comprising an amino acid sequence as set out in SEQ ID NO: 14 , aCDR2 comprising an amino acid sequence as set out in SEQ ID NO: 15 and aCDR3 comprising an amino acid sequence as set out in SEQ ID NO: 2,wherein the inflammatory disease is rheumatoid arthritis (RA), asthma,or multiple sclerosis (MS).
 12. A method of treating an inflammatorydisease, comprising administering an effective dose of an immunoglobulinG antibody which binds to human GM-CSF, wherein the antibody comprisesin its light chain variable region a CDR1 comprising an amino acidsequence as set out in SEQ ID NO: 16, a CDR2 comprising an amino acidsequence as set out in SEQ ID NO: 17 and a CDR3 comprising an amino acidsequence as set out in SEQ ID NO: 18; and comprising in its heavy chainvariable region a CDR1comprising an amino acid sequence as set out inSEQ ID NO: 14 , a CDR2 comprising an amino acid sequence as set out inSEQ ID NO: 15 and a CDR3 comprising an amino acid sequence as set out inSEQ ID NO: 2, wherein the inflammatory disease is rheumatoid arthritis(RA), asthma, or multiple sclerosis (MS).
 13. The method of claim 1,wherein the inflammatory disease is RA.
 14. The method of claim 13,wherein the RA is resistant to treatment with TNF-alpha neutralizers.15. The method of claim 1, wherein the inflammatory disease is asthma.16. The method of claim 1, wherein the inflammatory disease is MS. 17.The method of claim 11, wherein the inflammatory disease is RA.
 18. Themethod of claim 11, wherein the inflammatory disease is asthma.
 19. Themethod of claim 11, wherein the inflammatory disease is MS.